Methods of increasing the cellulolytic enhancing activity of a polypeptide

ABSTRACT

The present invention relates to methods of increasing the activity of a polypeptide having cellulolytic enhancing activity, comprising: adding a soluble activating divalent metal cation to a composition comprising the polypeptide having cellulolytic enhancing activity, wherein the presence of the soluble activating divalent metal cation and the polypeptide having cellulolytic enhancing activity increases degradation or conversion of a cellulose-containing material by a cellulolytic enzyme composition compared to the polypeptide having cellulolytic enhancing activity without the soluble activating divalent metal cation. The present invention also relates to compositions, methods for degrading or converting a cellulose-containing material, and methods for producing a fermentation product.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. ? filed on ?, which is a 35 U.S.C. §371 national application ofPCT/US2008/065360 filed on May 30, 2008 and claims priority from U.S.provisional application Ser. No. 60/941,243 filed on May 31, 2007, whichapplications are fully incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods and compositions for increasingthe activity of a polypeptide having cellulolytic enhancing activity.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

WO 2005/074647 discloses isolated polypeptides having cellulolyticenhancing activity and polynucleotides thereof from Thielaviaterrestris. WO 2005/074656 discloses an isolated polypeptide havingcellulolytic enhancing activity and a polynucleotide thereof fromThermoascus aurantiacus. U.S. Published Application Ser. No.2007/0077630 discloses an isolated polypeptide having cellulolyticenhancing activity and a polynucleotide thereof from Trichoderma reesei.

It would be an advantage in the art to improve the activity ofpolypeptides having cellulolytic enhancing activity.

The present invention relates to methods and compositions for increasingthe activity of a polypeptide having cellulolytic enhancing activity.

SUMMARY OF THE INVENTION

The present invention relates to methods of increasing the activity of apolypeptide having cellulolytic enhancing activity, comprising: adding asoluble activating divalent metal cation to a composition comprising thepolypeptide having cellulolytic enhancing activity, wherein the solubleactivating divalent metal cation is present at an effectiveconcentration of about 0.001 mM to about 50 mM during degradation orconversion of a cellulose-containing material and the presence of thesoluble activating divalent metal cation and the polypeptide havingcellulolytic enhancing activity increases the degradation or conversionof the cellulose-containing material by a cellulolytic enzymecomposition compared to the polypeptide having cellulolytic enhancingactivity without the soluble activating divalent metal cation.

The present invention also relates to methods for degrading orconverting a cellulose-containing material, comprising: treating thecellulose-containing material with an effective amount of a cellulolyticenzyme composition comprising an effective amount of a polypeptidehaving cellulolytic enhancing activity and a soluble activating divalentmetal cation, wherein the soluble activating divalent metal cation ispresent at an effective concentration of about 0.001 mM to about 50 mM.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying acellulose-containing material with an effective amount of a cellulolyticenzyme composition comprising an effective amount of a polypeptidehaving cellulolytic enhancing activity and a soluble activating divalentmetal cation, wherein the soluble activating divalent metal cation ispresent at an effective concentration of about 0.001 mM to about 50 mM;(b) fermenting the saccharified cellulose-containing material of step(a) with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to compositions comprising apolypeptide having cellulolytic enhancing activity and a solubleactivating divalent metal cation, wherein the soluble activatingdivalent metal cation is present at an effective concentration of about0.001 mM to about 50 mM during degradation or conversion of acellulose-containing material and the presence of the soluble activatingdivalent metal cation and the polypeptide having cellulolytic enhancingactivity increases the degradation or conversion of thecellulose-containing material by a cellulolytic enzyme compositioncompared to the polypeptide having cellulolytic enhancing activitywithout the soluble activating divalent metal cation.

The present invention also relates to cellulolytic enzyme compositionscomprising an effective amount of a polypeptide having cellulolyticenhancing activity and a soluble activating divalent metal cation,wherein the soluble activating divalent metal cation is present at aneffective concentration of about 0.001 mM to about 50 mM duringdegradation or conversion of a cellulose-containing material and thepresence of the soluble activating divalent metal cation and thepolypeptide having cellulolytic enhancing activity increases thedegradation or conversion of the cellulose-containing material by thecellulolytic enzyme compositions compared to the polypeptide havingcellulolytic enhancing activity without the soluble activating divalentmetal cation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pMJ04.

FIG. 2 shows a restriction map of pCaHj527.

FIG. 3 shows a restriction map of pMT2188.

FIG. 4 shows a restriction map of pCaHj568.

FIG. 5 shows a restriction map of pMJ05.

FIG. 6 shows a restriction map of pSMai130.

FIG. 7 shows the DNA sequence and amino acid sequence of an Aspergillusoryzae beta-glucosidase native signal sequence (SEQ ID NOs: 57 and 58).

FIG. 8 shows the the DNA sequence and amino acid sequence of a Humicolainsolens endoglucanase V signal sequence (SEQ ID NOs: 61 and 62).

FIG. 9 shows a restriction map of pSMai135.

FIG. 10 shows a restriction map of pSMai140.

FIG. 11 shows a restriction map of pSaMe-F1.

FIGS. 12A, 12B, 12C, and 12D show the DNA sequence and deduced aminoacid sequence of an Aspergillus oryzae beta-glucosidase variant BGfusion protein (SEQ ID NOs: 25 and 26, respectively).

FIG. 13 shows a restriction map of pSaMe-FX.

FIGS. 14A, 14B, 14C, and 14D show the DNA sequence and deduced aminoacid sequence of the Aspergillus oryzae beta-glucosidase fusion protein(SEQ ID NOs: 27 and 28, respectively).

FIG. 15 shows a restriction map of pAILo47.

FIG. 16 shows the conversion of cellulose in pretreated corn stover toglucose and cellobiose with addition of various soluble divalent metalions to a final concentration of 10 mM to mixtures including afermentation broth that comprises Trichoderma reesei cellulolyticenzymes, an Aspergillus oryzae beta-glucosidase fusion protein, and aThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity.

FIG. 17 shows the conversion of cellulose in pretreated corn stover toglucose and cellobiose with addition of various divalent metal ions to afinal concentration of 1 mM in mixtures including a fermentation broththat comprises Trichoderma reesei cellulolytic enzymes and anAspergillus oryzae beta-glucosidase with and without addition of aThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity.

FIG. 18 shows the conversion of cellulose in pretreated corn stover toglucose and cellobiose with addition of MgCl₂ and MnSO₄ to a finalconcentration of 0.0001 to 10 mM in mixtures including desalted orun-desalted fermentation broth that comprises Trichoderma reeseicellulolytic enzymes, an Aspergillus oryzae beta-glucosidase fusionprotein, and a Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity.

Definitions

Cellulolytic enhancing activity: The term “cellulolytic enhancingactivity” is defined herein as a biological activity that enhances thehydrolysis of a cellulose-containing material by proteins havingcellulolytic activity. For purposes of the present invention,cellulolytic enhancing activity is determined by measuring the increasein reducing sugars or the increase of the total of cellobiose andglucose from the hydrolysis of a cellulose-containing material bycellulolytic protein under the following conditions: 1-50 mg of totalprotein containing 80-99.5% w/w cellulolytic protein/g of cellulose inPCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1-7day at 50° C. compared to a control hydrolysis with equal total proteinloading without cellulolytic enhancing activity (1-50 mg of cellulolyticprotein/g of cellulose in PCS). In a preferred aspect, a mixture ofCELLUCLAST® 1.5 L (Novozymes A/S, Bagsværd, Denmark) in the presence of3% Aspergillus oryzae beta-glucosidase (recombinantly produced inAspergillus oryzae according to WO 02/095014) or 3% Aspergillusfumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzaeaccording to Example 22 of WO 02/095014) of cellulase protein loading isused as a standard of the cellulolytic activity.

The polypeptides having cellulolytic enhancing activity have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8,10, 12, or 14.

The polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulose-containing material catalyzed by proteinshaving cellulolytic activity by reducing the amount of cellulolyticenzyme required to reach the same degree of hydrolysis preferably atleast 0.1-fold, more at least 0.2-fold, more preferably at least0.3-fold, more preferably at least 0.4-fold, more preferably at least0.5-fold, more preferably at least 1-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, more preferably at least 10-fold, more preferably at least20-fold, even more preferably at least 30-fold, most preferably at least50-fold, and even most preferably at least 100-fold.

Cellulolytic activity: The term “cellulolytic activity” is definedherein as cellulase activity (e.g., endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof) thathydrolyzes a cellulose-containing material. Cellulolytic protein mayhydrolyze or hydrolyzes carboxymethyl cellulose (CMC), therebydecreasing the viscosity of the incubation mixture. The resultingreduction in viscosity may be determined by a vibration viscosimeter(e.g., MIVI 3000 from Sofraser, France). Determination of cellulaseactivity, measured in terms of Cellulase Viscosity Unit (CEVU),quantifies the amount of catalytic activity present in a sample bymeasuring the ability of the sample to reduce the viscosity of asolution of carboxymethyl cellulose (CMC). The assay is performed at thetemperature and pH suitable for the cellulolytic protein and substrate.

For purposes of the present invention, cellulolytic activity isdetermined by measuring the increase in hydrolysis of acellulose-containing material by a cellulolytic composition under thefollowing conditions: 1-50 mg of cellulolytic protein/g of cellulose inPCS for 1-7 day at 50° C. compared to a control hydrolysis withoutaddition of cellulolytic protein.

Endoglucanase: The term “endoglucanase” is defined herein as anendo-1,4-(1,3; 1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4),which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

Cellobiohydrolase: The term “cellobiohydrolase” is defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain. For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279 and by van Tilbeurgh et al., 1982,FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBSLetters 187: 283-288. In the present invention, the Lever et al. methodwas employed to assess hydrolysis of cellulose in corn stover, while themethod of van Tilbeurgh et al. was used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al., 2002, J. Basic Microbiol. 42: 55-66, exceptdifferent conditions were employed as described herein. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% TWEEN® 20.

Family 7, 12, 45, or 61 glycoside hydrolase: The term “Family 7glycoside hydrolase” or “Family GH7”, “Family 12 glycoside hydrolase” or“Family GH12”, “Family 45 glycoside hydrolase” or “Family GH45”, and“Family 61 glycoside hydrolase” or “Family GH61” is defined herein as apolypeptide falling into the glycoside hydrolase Family 7, Family 12,Family 45, and Family 61, respectively, according to Henrissat, 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. Presently, Henrissat lists theGH61 Family as unclassified indicating that properties such asmechanism, catalytic nucleophile/base, catalytic proton donors, and 3-Dstructure are not known for polypeptides belonging to this family. AGH7, GH12, or GH45 protein is also referred to as a CEL7, CEL12, orCEL45 protein, respectively.

Cellulose-containing material: The predominant polysaccharide in theprimary cell wall of biomass is cellulose, the second most abundant ishemi-cellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

The cellulose-containing material can be any material containingcellulose. Cellulose is generally found, for example, in the stems,leaves, hulls, husks, and cobs of plants or leaves, branches, and woodof trees. The cellulose-containing material can be, but is not limitedto, herbaceous material, agricultural residues, forestry residues,municipal solid wastes, waste paper, and pulp and paper mill residues.The cellulose-containing material can be any type of biomass including,but not limited to, wood resources, municipal solid waste, wastepaper,crops, and crop residues (see, for example, Wiselogel et al., 1995, inHandbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor &Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16;Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719;Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, NewYork). It is understood herein that the cellulose-containing material ispreferably in the form of lignocellulose, e.g., a plant cell wallmaterial containing lignin, cellulose, and hemicellulose in a mixedmatrix.

In a preferred aspect, the cellulose-containing material is corn stover.In another preferred aspect, the cellulose-containing material is cornfiber. In another preferred aspect, the cellulose-containing material iscorn cobs. In another preferred aspect, the cellulose-containingmaterial is switch grass. In another preferred aspect, thecellulose-containing material is rice straw. In another preferredaspect, the cellulose-containing material is paper and pulp processingwaste. In another preferred aspect, the cellulose-containing material iswoody or herbaceous plants. In another preferred aspect, thecellulose-containing material is bagasse.

The cellulose-containing material may be used as is or may be subjectedto pretreatment, using conventional methods known in the art. Forexample, physical pretreatment techniques can include various types ofmilling, irradiation, steaming/steam explosion, and hydrothermolysis;chemical pretreatment techniques can include dilute acid, alkaline,organic solvent, ammonia, sulfur dioxide, carbon dioxide, andpH-controlled hydrothermolysis; and biological pretreatment techniquescan involve applying lignin-solubilizing microorganisms (see, forexample, Hsu, T. -A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, DC, 179-212; Ghosh, P., and Singh, A., 1993,Physicochemical and biological treatments for enzymatic/microbialconversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39:295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: areview, in Enzymatic Conversion of Biomass for Fuels Production, Himmel,M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566,American Chemical Society, Washington, DC, chapter 15; Gong, C. S., Cao,N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewableresources, in Advances in Biochemical Engineering/Biotechnology,Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Olsson, L., and Hahn-Hagerdal, B., 1996, Fermentation oflignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech.18: 312-331; and Vallander, L., and Eriksson, K. -E. L., 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Pre-treated corn stover: The term “PCS” or “Pre-treated Corn Stover” isdefined herein as a cellulose-containing material derived from cornstover by treatment with heat and dilute acid. For purposes of thepresent invention, PCS is made by the method described in Example 20, orvariations thereof in time, temperature and amount of acid.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form, i.e.,that the polypeptide preparation is essentially free of otherpolypeptide material with which it is natively or recombinantlyassociated. This can be accomplished, for example, by preparing thepolypeptide by well-known recombinant methods or by classicalpurification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having biological activity, e.g., enzyme activity, which isin its final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having biological activity.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the—nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues ×100)/(Length of Alignment—Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBl NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment—Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein assequences with an E value (or expectancy score) of less than 0.001 usingthe blastp (for protein databases) or tblastn (for nucleic aciddatabases) algorithms with the BLOSUM62 matrix, wordsize 3, gapexistence cost 11, gap extension cost 1, no low complexity filtration,and a mature protein sequence as query. See Altschul et al., 1997,Nucleic Acids Res. 25: 3389-3402.

Polypeptide Fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of the mature polypeptide or ahomologous sequence thereof; wherein the fragment has activity as themature polypeptide thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of the mature polypeptide coding sequence or a homologoussequence thereof; wherein the subsequence encodes a polypeptide fragmenthaving activity as the mature polypeptide thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps before appearing as mature spliced mRNA. These steps includethe removal of intron sequences by a process called splicing. cDNAderived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide.

Modification: The term “modification” means herein any chemicalmodification of a mature polypeptide or a homologous sequence thereof;as well as genetic manipulation of the DNA encoding such a polypeptide.The modification can be a substitution, a deletion and/or an insertionof one or more (several) amino acids as well as replacements of one ormore (several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide produced by an organism expressing a modifiednucleotide sequence of a mature polypeptide coding sequence or ahomologous sequence thereof. The modified nucleotide sequence isobtained through human intervention by modification of thepolynucleotide sequence or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of increasing the activity of apolypeptide having cellulolytic enhancing activity, comprising: adding asoluble activating divalent metal cation to a composition comprising thepolypeptide having cellulolytic enhancing activity, wherein the solubleactivating divalent metal cation is present at an effectiveconcentration of about 0.001 mM to about 50 mM during degradation orconversion of a cellulose-containing material and the presence of thesoluble activating divalent metal cation and the polypeptide havingcellulolytic enhancing activity increases the degradation or conversionof the cellulose-containing material by a cellulolytic enzymecomposition compared to the polypeptide having cellulolytic enhancingactivity without the soluble activating divalent metal cation.

The present invention also relates to methods for degrading orconverting a cellulose-containing material, comprising: treating thecellulose-containing material with an effective amount of a cellulolyticenzyme composition comprising an effective amount of a polypeptidehaving cellulolytic enhancing activity and a soluble activating divalentmetal cation, wherein the soluble activating divalent metal cation ispresent at an effective concentration of about 0.001 mM to about 50 mM.

The present invention also relates to methods for producing afermentation product, comprising: (a) saccharifying acellulose-containing material with an effective amount of a cellulolyticenzyme composition comprising an effective amount of a polypeptidehaving cellulolytic enhancing activity and a soluble activating divalentmetal cation, wherein the soluble activating divalent metal cation ispresent at an effective concentration of about 0.001 mM to about 50 mM;(b) fermenting the saccharified cellulose-containing material of step(a) with one or more fermenting microorganisms to produce thefermentation product; and (c) recovering the fermentation product fromthe fermentation.

The present invention also relates to compositions comprising apolypeptide having cellulolytic enhancing activity and a solubleactivating divalent metal cation, wherein the soluble activatingdivalent metal cation is present at an effective concentration of about0.001 mM to about 50 mM during degradation or conversion of acellulose-containing material and the presence of the soluble activatingdivalent metal cation and the polypeptide having cellulolytic enhancingactivity increases the degradation or conversion of thecellulose-containing material by a cellulolytic enzyme compositioncompared to the polypeptide having cellulolytic enhancing activitywithout the soluble activating divalent metal cation.

The present invention also relates to cellulolytic enzyme compositionscomprising an effective amount of a polypeptide having cellulolyticenhancing activity and a soluble activating divalent metal cation,wherein the soluble activating divalent metal cation is present at aneffective concentration of about 0.001 mM to about 50 mM duringdegradation or conversion of a cellulose-containing material and thepresence of the soluble activating divalent metal cation and thepolypeptide having cellulolytic enhancing activity increases thedegradation or conversion of the cellulose-containing material by thecellulolytic enzyme compositions compared to the polypeptide havingcellulolytic enhancing activity without the soluble activating divalentmetal cation.

Divalent Metal Cations

Any soluble activating divalent metal cation can be used in the presentinvention. In a preferred aspect, the soluble activating divalent metalcation is selected from the group consisting of Mn⁺⁺, Co⁺⁺, Mg⁺⁺, Ca⁺⁺,and a combination thereof. In a more preferred aspect, the solubleactivating divalent metal cation isMn^(++. In another more preferred aspect, the soluble activating divalent metal cation is Co)⁺⁺. In another more preferred aspect, the soluble activating divalentmetal cation is Mg⁺⁺. In another more preferred aspect, the solubleactivating divalent metal cation is Ca⁺⁺. In another more preferredaspect, the soluble activating divalent metal cation is two or more(several) cations selected from the group consisting of Mn⁺⁺, Co⁺⁺,Mg⁺⁺, and Ca⁺⁺. In a most preferred aspect, the soluble activatingdivalent metal cation is Mn⁺⁺. In another most preferred aspect, thesoluble activating divalent metal cation is Mg^(++.)

The soluble activating divalent metal cation is added during thedegradation or conversion of a cellulose-containing substrate at aneffective concentration of preferably about 0.001 mM to about 50 mM,more preferably about 0.01 mM to about 25 mM, more preferably about 0.1mM to about 25 mM, more preferably about 0.1 mM to about 10 mM, evenmore preferably about 0.3 mM to about 5 mM, most preferably about 0.3 mMto about 2.5 mM, and even most preferably about 0.3 mM to about 1 mM.

In a preferred aspect, the soluble activating divalent metal cation isadded during the degradation or conversion of a cellulose-containingsubstrate at an effective concentration of about 0.001 mM to about 50mM. In a more preferred aspect, the soluble activating divalent metalcation is added during the degradation or conversion of acellulose-containing substrate at an effective concentration of about0.01 mM to about 25 mM. In a more preferred aspect, the solubleactivating divalent metal cation is added during the degradation orconversion of a cellulose-containing substrate at an effectiveconcentration of about 0.1 mM to about 25 mM. In a more preferredaspect, the soluble activating divalent metal cation is added during thedegradation or conversion of a cellulose-containing substrate at aneffective concentration of about 0.1 mM to about 10 mM. In a even morepreferred aspect, the soluble activating divalent metal cation is addedduring the degradation or conversion of a cellulose-containing substrateat an effective concentration of about 0.3 mM to about 5 mM. In a mostpreferred aspect, the soluble activating divalent metal cation is addedduring the degradation or conversion of a cellulose-containing substrateat an effective concentration of about 0.3 mM to about 2.5 mM. In a evenmost preferred aspect, the soluble activating divalent metal cation isadded during the degradation or conversion of a cellulose-containingsubstrate at an effective concentration of about 0.3 mM to about 1 mM.

The soluble activating divalent metal cation is preferably added as asoluble salt, such as, for example, a sulfate, carbonate, chloride,citrate, nitrate, nitrite, fluoride, or iodide salt. It is well known inthe art, however, that cellulosic biomass can comprise a number ofdivalent metal cations. See, for example, F. B. Salisbury and C. W.Ross: Plant Physiology, Wadsworths Publishing Company, Belmont, Calif.(1992). The cellulosic biomass can, therefore, be used, in part orwholly, as a source of the metal cations. The activating divalent metalcations may be soluble or insoluble. The term “soluble activatingdivalent metal cation” is defined herein as a divalent metal cation thatis available in solution to increase the activity of a polypeptidehaving cellulolytic enhancing activity. The term “insoluble activatingdivalent metal cation” is defined herein as a divalent metal cation thatis unavailable in solution to increase the activity of a polypeptidehaving cellulolytic enhancing activity. The divalent metal cation may beunavailable because, for example, it is chelated by, for example, EDTAor EGTA, or it is complexed with a component of the cellulosic biomass,for example, pyrophosphate.

The cellulosic biomass can also supply soluble divalent metal cationsthat inhibit the cellulolysis (hereinafter “soluble inhibitory divalentmetal cation”). For example, an inhibitory divalent metal cation isZn⁺⁺orFe^(++. Consequently, under conditions where a mixture of soluble divalent metal cations are present, some that activate and others that inhibit cellulolysis, an excess of a soluble activating divalent metal cation is added to overcome the inhibitory effect of the inhibitory divalent metal cations. In such a situation to prevent inhibitory divalent metal cations from adversely affecting the polypeptide having cellulolytic enhancing activity, the methods of the present invention further comprise supplementing the concentration of the soluble activating divalent metal cation to maintain the effective concentration of the soluble activating divalent metal cation in the range of preferably about)0.001 mM to about 50 mM, more preferably about 0.01 mM to about 25 mM,more preferably about 0.1 mM to about 25 mM, more preferably about 0.1mM to about 10 mM, even more preferably about 0.3 mM to about 5 mM, mostpreferably about 0.3 mM to about 2.5 mM, and even most preferably about0.3 mM to about 1 mM for a period sufficient to degrade or convert thecellulose-containing material.

In a preferred aspect, the concentration of the soluble activatingdivalent metal cation is supplemented during the degradation orconversion of a cellulose-containing substrate to maintain an effectiveconcentration of about 0.001 mM to about 50 mM. In a more preferredaspect, the concentration of the soluble activating divalent metalcation is supplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.01 mM to about 25 mM. In a more preferred aspect, theconcentration of the soluble activating divalent metal cation issupplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.1 mM to about 25 mM. In a more preferred aspect, theconcentration of the soluble activating divalent metal cation issupplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.1 mM to about 10 mM. In a even more preferred aspect, theconcentration of the soluble activating divalent metal cation issupplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.3 mM to about 5 mM. In a most preferred aspect, theconcentration of the soluble activating divalent metal cation issupplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.3 mM to about 2.5 mM. In a even most preferred aspect, theconcentration of the soluble activating divalent metal cation issupplemented during the degradation or conversion of acellulose-containing substrate to maintain an effective concentration ofabout 0.3 mM to about 1 mM.

The concentration of divalent metal cations in cellulosic biomass can bedetermined using any method known in the art, such as atomic absorption,electrochemical electrodes, metal ion biosensors, or optical sensors, ortitration by chelation (see, for example, Methods in Enzymology, v.158(multiple chapters), Haugland, R. P. Handbook of Fluorescent Probes andResearch Chemicals, 6th ed.; Molecular Probes, Inc.: Eugene, Oreg.,1996., Thompson et al. Anal. Chem., 70 (22), 4717-4723, 1998,Inductively Coupled Plasma Mass Spectrometry, Akbar Montaser (Editor)May 1998).

In the methods of the present invention, a soluble activating divalentmetal cation increases the activity of a polypeptide having cellulolyticenhancing activity preferably at least 0.1-fold, more preferably atleast 0.2-fold, more preferably at least 0.3-fold, more preferably atleast 0.4-fold, more preferably at least 0.5-fold, more preferably atleast 1-fold, more preferably at least 3-fold, more preferably at least4-fold, more preferably at least 5-fold, more preferably at least10-fold, more preferably at least 20-fold, even more preferably at least30-fold, most preferably at least 50-fold, and even most preferably atleast 100-fold.

Cellulolytic Enzyme Compositions

The present invention also relates to compositions comprising apolypeptide having cellulolytic enhancing activity and a solubleactivating divalent metal cation, wherein the soluble activatingdivalent metal cation is present at an effective concentration ofpreferably about 0.001 mM to about 50 mM, more preferably about 0.01 mMto about 25 mM, more preferably about 0.1 mM to about 25 mM, morepreferably about 0.1 mM to about 10 mM, even more preferably about 0.3mM to about 5 mM, most preferably about 0.3 mM to about 2.5 mM, and evenmost preferably about 0.3 mM to about 1 mM during degradation orconversion of a cellulose-containing material and the presence of thesoluble activating divalent metal cation and the polypeptide havingcellulolytic enhancing activity increases the degradation or conversionof the cellulose-containing material by a cellulolytic enzymecomposition compared to the polypeptide having cellulolytic enhancingactivity without the soluble activating divalent metal cation.

The present invention also relates to cellulolytic enzyme compositionscomprising an effective amount of a polypeptide having cellulolyticenhancing activity and a soluble activating divalent metal cation,wherein the soluble activating divalent metal cation is present at aneffective concentration of preferably about 0.001 mM to about 50 mM,more preferably about 0.01 mM to about 25 mM, more preferably about 0.1mM to about 25 mM, more preferably about 0.1 mM to about 10 mM, evenmore preferably about 0.3 mM to about 5 mM, most preferably about 0.3 mMto about 2.5 mM, and even most preferably about 0.3 mM to about 1 mMduring degradation or conversion of a cellulose-containing material andthe presence of the soluble activating divalent metal cation and thepolypeptide having cellulolytic enhancing activity increases thedegradation or conversion of the cellulose-containing material by thecellulolytic enzyme compositions compared to the polypeptide havingcellulolytic enhancing activity without the soluble activating divalentmetal cation.

In the methods of the present invention, the cellulolytic enzymecomposition may comprise any protein involved in the processing of acellulose-containing material to glucose, or hemicellulose to xylose,mannose, galactose, and arabinose, their polymers, or products derivedfrom them as described below. In one aspect, the cellulolytic enzymecomposition comprises an endoglucanase, a cellobiohydrolase, abeta-glucosidase, or a combination thereof. In another aspect, thecellulolytic enzyme composition further comprises one or more additionalenzyme activities to improve the degradation of the cellulose-containingmaterial. Preferred additional enzymes are hemicellulases, esterases(e.g., lipases, phospholipases, and/or cutinases), proteases, laccases,peroxidases, or mixtures thereof.

The cellulolytic enzyme composition may be a monocomponent preparation,e.g., an endoglucanase, a multicomponent preparation, e.g.,endoglucanase(s), cellobiohydrolase(s), and beta-glucosidase(s), or acombination of multicomponent and monocomponent protein preparations.The cellulolytic proteins may have activity, i.e., hydrolyze thecellulose-containing material, either in the acid, neutral, or alkalinepH-range.

As mentioned above, the cellulolytic proteins used in the presentinvention may be monocomponent preparations, i.e., a componentessentially free of other cellulolytic components. The single componentmay be a recombinant component, i.e., produced by cloning of a DNAsequence encoding the single component and subsequent cell transformedwith the DNA sequence and expressed in a host (see, for example, WO91/17243 and WO 91/17244). The host cell may be a heterologous host(enzyme is foreign to host) or the host may also be a wild-type host(enzyme is native to host). Monocomponent cellulolytic proteins may alsobe prepared by purifying such a protein from a fermentation broth.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

A polypeptide having cellulolytic enzyme activity may be obtained frommicroorganisms of any genus. The term “obtained from” means herein thatthe enzyme may have been isolated from an organism that naturallyproduces the enzyme as a native enzyme. The term “obtained from” alsomeans herein that the enzyme may have been produced recombinantly in ahost organism, wherein the recombinantly produced enzyme is eithernative or foreign to the host organism or has a modified amino acidsequence, e.g., having one or more amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtained bychemical or recombinant mutagenesis, such as by site-directedmutagenesis or shuffling. Consequently, chemically modified or proteinengineered mutants of cellulolytic proteins may also be used in thepresent invention. In a preferred aspect, the polypeptide obtained froma given source is secreted extracellularly.

A polypeptide having cellulolytic enzyme activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enzymeactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enzyme activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enzyme activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenzyme activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enzyme activity.

The polypeptide having cellulolytic enzyme activity may also be a fungalpolypeptide, and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having cellulolytic enzyme activity; or more preferably afilamentous fungal polypeptide such as aan Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having cellulolyticenzyme activity.

In a preferred aspect, the polypeptide is a Saccharomycescarisbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenzyme activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, lrpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enzyme activity.

In the methods of the present invention, any endoglucanase,cellobiohydrolase, and/or beta-glucosidase, as well as any othercellulolytic enzyme, can be used.

Examples of bacterial endoglucanases that can be used in the presentinvention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GenBank™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo etal., 1988, Gene 63:11-22; GenBank™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.64: 555-563; GenBank™ accession no. AB003694); Trichoderma reeseiendoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591;GenBank™ accession no. Y11113); and Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GenBank™accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al.,1990, Nucleic Acids Research 18: 5884); Aspergillus kawachiiendoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439);Chrysosporium sp. C1 (U.S. Pat. No. 6,573,086; GenPept accession no.AAQ38150); Corynascus heterothallicus (U.S. Pat. No. 6,855,531; GenPeptaccession no. AAY00844); Erwinia carotovara endoglucanase (Saarilahti etal., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GenBank™accession no. L29381); Humicola grisea var. thermoidea endoglucanase(GenBank™ accession no. AB003107); Melanocarpus albomyces endoglucanase(GenBank™ accession no. MAL515703); Neurospora crassa endoglucanase(GenBank™ accession no. XM_324477); Piromyces equi (Eberhardt et al.,2000, Microbiology 146: 1999-2008; GenPept accession no. CAB92325);Rhizopus oryzae (Moriya et al., 2003, J. Bacteriology 185: 1749-1756;GenBank™ accession nos. AB047927, AB056667, and AB056668); and Thielaviaterrestris (WO 2004/053039; EMBL accession no. CQ827970).

Other endoglucanases are disclosed in more than 13 of the GlycosylHydrolase families using the classification according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino-acidsequence similarities, Biochem. J. 280: 309-316, and Henrissat B., andBairoch A., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

In a preferred aspect, the endoglucanase is a Trichoderma reeseiendoglucanase I (CEL7B). In another preferred aspect, the endoglucanaseis a Trichoderma reesei endoglucanase II (CEL5A). In another preferredaspect, the endoglucanase is a Trichoderma reesei endoglucanase III(CEL12A). In another preferred aspect, the endoglucanase is aTrichoderma reesei endoglucanase V (CEL45A). In another preferredaspect, the endoglucanase is a Myceliophthora thermophila CEL7endoglucanase. In another preferred aspect, the endoglucanase is aChrysosporium lucknowense CEL12 endoglucanase. In another preferredaspect, the endoglucanase is a Chrysosporium lucknowense CEL45endoglucanase.

In a more preferred aspect, the Trichoderma reesei endoglucanase I(CEL7B) is the mature polypeptide of SEQ ID NO: 74 or an ortholog orvariant thereof. In another more preferred aspect, the Trichodermareesei endoglucanase II (CEL5A) is the mature polypeptide of SEQ ID NO:76 or an ortholog or variant thereof. In another more preferred aspect,the Trichoderma reesei endoglucanase III (CEL12A) is the maturepolypeptide of SEQ ID NO: 78 or an ortholog or variant thereof. Inanother more preferred aspect, the Trichoderma reesei endoglucanase V(CEL45A) is the mature polypeptide of SEQ ID NO: 80 or an ortholog orvariant thereof. In another more preferred aspect, the Myceliophthorathermophila CEL7 endoglucanase is the mature polypeptide of SEQ ID NO:82 or an ortholog or variant thereof. In another more preferred aspect,the Myceliophthora thermophila CEL12 endoglucanase is the maturepolypeptide of SEQ ID NO: 84 or an ortholog or variant thereof. Inanother more preferred aspect, the Myceliophthora thermophila CEL45endoglucanase is the mature polypeptide of SEQ ID NO: 86 or an orthologor variant thereof.

In another more preferred aspect, the Trichoderma reesei endoglucanase I(CEL7B) is encoded by the mature polypeptide coding sequence of SEQ IDNO: 73 or an ortholog or variant thereof. In another more preferredaspect, the Trichoderma reesei endoglucanase II (CEL5A) is encoded bythe mature polypeptide coding sequence of SEQ ID NO: 75 or an orthologor variant thereof. In another more preferred aspect, the Trichodermareesei endoglucanase III (CEL12A) is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 77 or an ortholog or variant thereof. Inanother more preferred aspect, the Trichoderma reesei endoglucanase V(CEL45A) is encoded by the mature polypeptide coding sequence of SEQ IDNO: 79 or an ortholog or variant thereof. In another more preferredaspect, the Myceliophthora thermophila CEL7 endoglucanase is encoded bythe mature polypeptide coding sequence of SEQ ID NO: 81 or an orthologor variant thereof. In another more preferred aspect, the ChrysosporiumCEL12 endoglucanase is encoded by the mature polypeptide coding sequenceof SEQ ID NO: 83 or an ortholog or variant thereof. In another morepreferred aspect, the Chrysosporium CEL45 endoglucanase is encoded bythe mature polypeptide coding sequence of SEQ ID NO: 85 or an orthologor variant thereof.

The Trichoderma reesei endoglucanase I (CEL7B) can be obtained accordingto Penttila et al., 1986, Gene 45: 253-263. The Trichoderma reeseiendoglucanase II (CEL5A) can be obtained according to Saloheimo et al.,1988, Gene 63:11-22. The Trichoderma reesei endoglucanase III (CEL12A)can be obtained according to Okada et al., 1988, Appl. Environ.Microbiol. 64: 555-563. The Trichoderma reesei endoglucanase V (CEL45A)can be obtained according to Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228. The Myceliophthora thermophila CEL7endoglucanase can be obtained according to WO 95/024471. TheChrysosporium lucknowense CEL12 endoglucanase can be obtained accordingto WO 2001/25468. The Chrysosporium lucknowense CEL45 endoglucanase canbe obtained according to WO 2000/20555.

In another preferred aspect, the cellobiohydrolase is a Trichodermareesei cellobiohydrolase I (CEL7A). In another preferred aspect, thecellobiohydrolase is a Trichoderma reesei cellobiohydrolase II (CEL6A).In another preferred aspect, the cellobiohydrolase is a Chrysosporiumlucknowense CEL7 cellobiohydrolase with a cellulose binding domain. Inanother preferred aspect, the cellobiohydrolase is a Myceliophthorathermophila CEL7 cellobiohydrolase without a cellulose binding domain.In another preferred aspect, the cellobiohydrolase is a Thielaviaterrestris cellobiohydrolase.

In another more preferred aspect, the Trichoderma reeseicellobiohydrolase I (CEL7A) is the mature polypeptide of SEQ ID NO: 88or an ortholog or variant thereof. In another preferred aspect, theTrichoderma reesei cellobiohydrolase II (CEL6A) is the maturepolypeptide of SEQ ID NO: 90 or an ortholog or variant thereof. Inanother more preferred aspect, the Chrysosporium lucknowense CEL7cellobiohydrolase with a cellulose binding domain is the maturepolypeptide of SEQ ID NO: 92 or an ortholog or variant thereof. Inanother more preferred aspect, the Myceliophthora thermophila CEL7cellobiohydrolase without a cellulose binding domain is the maturepolypeptide of SEQ ID NO: 94 or an ortholog or variant thereof. Inanother more preferred aspect, the Thielavia terrestriscellobiohydrolase is the mature polypeptide of SEQ ID NO: 96 or anortholog or variant thereof.

In another more preferred aspect, the Trichoderma reeseicellobiohydrolase I (CEL7A) cellobiohydrolase is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 87 or an ortholog or variantthereof. In another more preferred aspect, the Trichoderma reeseicellobiohydrolase II (CEL6A) cellobiohydrolase is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 89 or an ortholog or variantthereof. In another more preferred aspect, the Chrysosporium lucknowenseCEL7 cellobiohydrolase with a cellulose binding domain is encoded by themature polypeptide coding sequence of SEQ ID NO: 91 or an ortholog orvariant thereof. In another more preferred aspect, the Myceliophthorathermophila CEL7 cellobiohydrolase without a cellulose binding domain isencoded by the mature polypeptide coding sequence of SEQ ID NO: 93 or anortholog or variant thereof. In another more preferred aspect, theThielavia terrestris cellobiohydrolase is encoded by the maturepolypeptide coding sequence of SEQ ID NO: 95 or an ortholog or variantthereof.

The Trichoderma reesei cellobiohydrolase I (CEL7A) can be obtainedaccording to Shoemaker et al., 1983, Biotechnology (N.Y.) 1: 691-696.The Trichoderma reesei cellobiohydrolase II (CEL6A) can be obtainedaccording to Terri et al., 1987, Gene 51: 43-52. The Chrysosporiumlucknowense CEL7 cellobiohydrolase with a cellulose binding domain canbe obtained according to WO 2001/79507. The Myceliophthora thermophilaCEL7 cellobiohydrolase without a cellulose binding domain can beobtained according to WO 2003/000941. The Thielavia terrestriscellobiohydrolase can be obtained according to WO 2006/074435.

In another preferred aspect, the beta-glucosidase is obtained fromAspergillus oryzae. In another preferred aspect, the beta-glucosidase isobtained from Aspergillus fumigatus. In another preferred aspect, thebeta-glucosidase is obtained from Penicillium brasilianum, e.g.,Penicillium brasilianum strain IBT 20888. In another preferred aspect,the beta-glucosidase is obtained from Aspergillus niger. In anotherpreferred aspect, the beta-glucosidase is obtained from Aspergillusaculeatus.

In a more preferred aspect, the Aspergillus oryzae beta-glucosidase isthe mature polypeptide of SEQ ID NO: 16 or an ortholog or variantthereof. In another more preferred aspect, the Aspergillus fumigatusbeta-glucosidase is the mature polypeptide of SEQ ID NO: 18 or anortholog or variant thereof. In another more preferred aspect, thePenicillium brasilianum beta-glucosidase is the mature polypeptide ofSEQ ID NO: 20 or an ortholog or variant thereof. In another morepreferred aspect, the Aspergillus niger beta-glucosidase is the maturepolypeptide of SEQ ID NO: 22 or an ortholog or variant thereof. Inanother more preferred aspect, the Aspergillus aculeatusbeta-glucosidase is the mature polypeptide of SEQ ID NO: 24 or anortholog or variant thereof. In another more preferred aspect, theAspergillus oryzae beta-glucosidase is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 15 or an ortholog or variant thereof. Inanother more preferred aspect, the Aspergillus fumigatusbeta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 17 or an ortholog or variant thereof. In another morepreferred aspect, the Penicillium brasilianum beta-glucosidase isencoded by the mature polypeptide coding sequence of SEQ ID NO: 19 or anortholog or variant thereof. In another more preferred aspect, theAspergillus niger beta-glucosidase is encoded by the mature polypeptidecoding sequence of SEQ ID NO: 21 or an ortholog or variant thereof. Inanother more preferred aspect, the Aspergillus aculeatusbeta-glucosidase is encoded by the mature polypeptide coding sequence ofSEQ ID NO: 23 or an ortholog or variant thereof.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

In another preferred aspect, the mature polypeptide of SEQ ID NO: 16 isencoded by a polynucleotide contained in the plasmid which is containedin E. coli DSM 14240. In another preferred aspect, the maturepolypeptide of SEQ ID NO: 18 is encoded by the polynucleotide containedin plasmid pEJG113 which is contained in E. coli NRRL B-30695. Inanother preferred aspect, the mature polypeptide of SEQ ID NO: 20 isencoded by a polynucleotide contained in plasmid pKKAB which iscontained in E. coli NRRL B-30860.

In another preferred aspect, the beta-glucosidase is the Aspergillusoryzae beta-glucosidase variant BG fusion protein of SEQ ID NO: 26. Inanother preferred aspect, the Aspergillus oryzae beta-glucosidasevariant BG fusion protein is encoded by the polynucleotide of SEQ ID NO:25. In another preferred aspect, the beta-glucosidase is the Aspergillusoryzae beta-glucosidase fusion protein of SEQ ID NO: 28. In anotherpreferred aspect, the Aspergillus oryzae beta-glucosidase fusion proteinis encoded by the polynucleotide of SEQ ID NO: 27.

Examples of other beta-glucosidases that can be used in the presentinvention, include, but are not limited to, an Aspergillus oryzaebeta-glucosidase (WO 02/095014; WO 04/099228); Aspergillus aculeatusbeta-glucosidase (Kawaguchi et al., 1996, Gene 173: 287-288);Aspergillus avenaceus beta-glucosidase (GenBank™ accession no.AY943971); Aspergillus fumigatus beta-glucosidase (GenBank™ accessionno. XM745234); Aspergillus kawachii beta-glucosidase (GenBank™ accessionno. AB003470); Aspergillus niger beta-glucosidase (GenBank™ AJ132386);Magnaporthe grisea beta-glucosidase (GenBank™ accession no. AY849670);Phanerochaete chrysosporium beta-glucosidase (GenBank™ accession no.AB253327); Talaromyces emersonii beta-glucosidase (GenBank™ accessionno. AY072918), and Trichoderma reesei beta-glucosidase (GenBank™accession nos. U09580, AB003110, AY281374, AY281375, AY281377, AY281378,and AY281379). Variants of beta-glucosidases may also be used such asthose described in WO 04/099228.

Other beta-glucosidases are disclosed in more than 13 of the GlycosylHydrolase families using the classification according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino-acidsequence similarities, Biochem. J. 280: 309-316, and Henrissat B., andBairoch A., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Furthermore, such polypeptides may beidentified and obtained from other sources including microorganismsisolated from nature (e.g., soil, composts, water, etc.) using theabove-mentioned probes. Techniques for isolating microorganisms fromnatural habitats are well known in the art. The polynucleotide may thenbe obtained by similarly screening a genomic or cDNA library of such amicroorganism. Once a polynucleotide sequence encoding a polypeptide hasbeen detected with the probe(s), the polynucleotide can be isolated orcloned by utilizing techniques that are well known to those of ordinaryskill in the art (see, e.g., Sambrook et al., 1989, supra).

Polypeptides having cellulolytic enzyme activity also include fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof having cellulolytic enzyme activity. Afused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) encoding a polypeptide having cellulolytic enzymeactivity. Techniques for producing fusion polypeptides are known in theart, and include ligating the coding sequences encoding the polypeptidesso that they are in frame and that expression of the fused polypeptideis under control of the same promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having cellulolytic enzyme activity from the fusion protein.Examples of cleavage sites include, but are not limited to, a Kex2 sitethat encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

In a preferred aspect, the cellulolytic enzyme composition comprises abeta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), aTrichoderma reesei cellobiohydrolase II (CEL6A), and a Trichodermareesei endoglucanase I (CEL7B).

In another preferred aspect, the cellulolytic enzyme compositioncomprises a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I(CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and aTrichoderma reesei endoglucanase I (CEL7B), and further comprises one ormore enzymes selected from the group consisting of a Trichoderma reeseiendoglucanase II (CEL5A), a Trichoderma reesei endoglucanase V (CEL45A),and a Trichoderma reesei endoglucanase III (CEL12A).

In another preferred aspect, the cellulolytic enzyme compositioncomprises a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I(CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and aTrichoderma reesei endoglucanase I (CEL7B), and further comprises aThielavia terrestris cellobiohydrolase.

In another preferred aspect, the cellulolytic enzyme compositioncomprises a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I(CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and aTrichoderma reesei endoglucanase I (CEL7B), and further comprises (1)one or more enzymes selected from the group consisting of a Trichodermareesei endoglucanase II (CEL5A), a Trichoderma reesei endoglucanase V(CEL45A), and a Trichoderma reesei endoglucanase III (CEL12A), and/orfurther comprises (2) a Thielavia terrestris cellobiohydrolase.

In another preferred aspect, the cellulolytic enzyme compositioncomprises one or more (several) components selected from the groupconsisting of a Myceliophthora thermophila CEL7 polypeptide havingendoglucanase activity, a Chrysosporium lucknowense CEL12 polypeptidehaving endoglucanase activity, a Chrysosporium lucknowense CEL45polypeptide having endoglucanase activity, a Chrysosporium lucknowenseCEL7 polypeptide having cellobiohydrolase activity with a cellulosebinding domain, and a Myceliophthora thermophila CEL7 polypeptide havingcellobiohydrolase activity without a cellulose binding domain. Inanother preferred aspect, the cellulolytic enzyme composition comprisesa Myceliophthora thermophila CEL7 polypeptide having endoglucanaseactivity, a Chrysosporium lucknowense CEL12 polypeptide havingendoglucanase activity, a Chrysosporium lucknowense CEL45 polypeptidehaving endoglucanase activity, a CEL7 polypeptide havingcellobiohydrolase activity with a cellulose binding domain, and aMyceliophthora thermophila CEL7 polypeptide having cellobiohydrolaseactivity without a cellulose binding domain. In another preferredaspect, the composition above further comprises one or more (several)polypeptides having beta-glucosidase activity.

The cellulolytic enzyme composition can also be a commercialpreparation. Examples of commercial cellulolytic enzyme preparationssuitable for use in the present invention include, for example,CELLUCLAST™ (available from Novozymes A/S) and NOVOZYM™ 188 (availablefrom Novozymes A/S). Other commercially available preparations that maybe used include CELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S),LAMINEX™ and SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (Rohm GmbH),and FIBREZYME® LDI, FIBREZYME® LBR, or VISCOSTAR® 150 L (DyadicInternational, Inc., Jupiter, Fla., USA).

Other cellulolytic proteins that may be useful in the present inventionare described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

The cellulolytic proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, CA, 1991). Suitable media are available from commercial suppliersor may be prepared according to published compositions (e.g., incatalogues of the American Type Culture Collection). Temperature rangesand other conditions suitable for growth and cellulolytic proteinproduction are known in the art (see, e.g., Bailey, J. E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company,N.Y., 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe cellulolytic protein to be expressed or isolated. The resultingcellulolytic proteins produced by the methods described above may berecovered from the fermentation medium and purified by conventionalprocedures as described herein.

Polypeptides Having Cellulolytic Enhancing Activity

In a first aspect, the isolated polypeptides having cellulolyticenhancing activity comprise the following motifs:

(SEQ ID NO: 97 or SEQ ID NO: 98)[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and (SEQ ID NO: 104),[FW]-[TF]-K-[AIV]wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The isolated polypeptide comprising the above-noted motifs may furthercomprise:

(SEQ ID NO: 99 or SEQ ID NO: 100) H-X(1,2)-G-P-X(3)-[YW]-[AILMV], (SEQID NO: 101) [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or (SEQ ID NO: 99or SEQ ID NO: 100) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and (SEQ ID NO: 101)[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the isolated polypeptide having cellulolyticenhancing activity further comprises H—X(1,2)-G-P—X(3)-[YW]-[AILMV] (SEQID NO: 99 or SEQ ID NO: 100). In another preferred aspect, the isolatedpolypeptide having cellulolytic enhancing activity further comprises[EQ]—X—Y—X(2)—C—X-[EHQN]-[FILV]-X-[ILV] (SEQ ID NO: 101). In anotherpreferred aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises H—X(1,2)-G-P—X(3)-[YW]-[AILMV] (SEQ ID NO: 99or SEQ ID NO: 100) and [EQ]-X—Y—X(2)—C—X-[EHQN]-[FILV]—X-[ILV] (SEQ IDNO: 101). In a second aspect, the isolated polypeptides havingcellulolytic enhancing activity comprise the following motif:

(SEQ ID NO: 102 or SEQ ID NO: 103)[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)- A-[HNQ],wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

In a third aspect, the isolated polypeptides having cellulolyticenhancing activity have an amino acid sequence that has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, 97%, 98%, or 99%, which have cellulolytic enhancing activity(hereinafter “homologous polypeptides”). In a preferred aspect, thehomologous polypeptides have an amino acid sequence that differs by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 2. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 20 to 326 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 326 of SEQ ID NO: 2. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 20 to 326 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 326 of SEQ ID NO: 2.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 4 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 4. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises amino acids 18 to 240 of SEQ ID NO: 4, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 18 to 240 of SEQ ID NO: 4. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 4 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide consists of amino acids 18 to 240 of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 18 to 240 of SEQ ID NO: 4.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 6 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 6. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide comprises amino acids 20 to 258 of SEQ ID NO: 6, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 258 of SEQ ID NO: 6. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 6 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide consists of amino acids 20 to 258 of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 258 of SEQ ID NO: 6.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 8 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 8. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide comprises amino acids 19 to 226 of SEQ ID NO: 8, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 19 to 226 of SEQ ID NO: 8. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 8 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 8. In another preferred aspect, thepolypeptide consists of amino acids 19 to 226 of SEQ ID NO: 8 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 19 to 226 of SEQ ID NO: 8.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 10 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 10. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 10. In another preferred aspect,the polypeptide comprises amino acids 20 to 304 of SEQ ID NO: 10, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 304 of SEQ ID NO: 10. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 10 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 10. In another preferred aspect, thepolypeptide consists of amino acids 20 to 304 of SEQ ID NO: 10 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 304 of SEQ ID NO: 10.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 12 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 12. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 12. In another preferred aspect,the polypeptide comprises amino acids 23 to 250 of SEQ ID NO: 12, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 23 to 250 of SEQ ID NO: 12. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 12 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 12. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 12. In another preferred aspect, thepolypeptide consists of amino acids 23 to 250 of SEQ ID NO: 12 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 23 to 250 of SEQ ID NO: 12.

A polypeptide having cellulolytic enhancing activity preferablycomprises the amino acid sequence of SEQ ID NO: 14 or an allelic variantthereof; or a fragment thereof that has cellulolytic enhancing activity.In a preferred aspect, the polypeptide comprises the amino acid sequenceof SEQ ID NO: 14. In another preferred aspect, the polypeptide comprisesthe mature polypeptide of SEQ ID NO: 14. In another preferred aspect,the polypeptide comprises amino acids 20 to 249 of SEQ ID NO: 14, or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 20 to 249 of SEQ ID NO: 14. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 14 or an allelic variant thereof; or a fragment thereof that hascellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 14. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 14. In another preferred aspect, thepolypeptide consists of amino acids 20 to 249 of SEQ ID NO: 14 or anallelic variant thereof; or a fragment thereof that has cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 20 to 249 of SEQ ID NO: 14.

Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2contains at least 277 amino acid residues, more preferably at least 287amino acid residues, and most preferably at least 297 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:4 contains at least 185 amino acid residues, more preferably at least195 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:6 contains at least 200 amino acid residues, more preferably at least212 amino acid residues, and most preferably at least 224 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:8 contains at least 175 amino acid residues, more preferably at least185 amino acid residues, and most preferably at least 195 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:10 contains at least 240 amino acid residues, more preferably at least255 amino acid residues, and most preferably at least 270 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:12 contains at least 175 amino acid residues, more preferably at least190 amino acid residues, and most preferably at least 205 amino acidresidues. Preferably, a fragment of the mature polypeptide of SEQ ID NO:14 contains at least 200 amino acid residues, more preferably at least210 amino acid residues, and most preferably at least 220 amino acidresidues.

Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least861 nucleotides, and most preferably at least 891 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least585 nucleotides, and most preferably at least 615 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least636 nucleotides, and most preferably at least 672 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least555 nucleotides, and most preferably at least 585 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofSEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least765 nucleotides, and most preferably at least 810 nucleotides.Preferably, a subsequence of the mature polypeptide coding sequence ofnucleotides 67 to 796 of SEQ ID NO: 11 contains at least 525nucleotides, more preferably at least 570 nucleotides, and mostpreferably at least 615 nucleotides. Preferably, a subsequence of themature polypeptide coding sequence of SEQ ID NO: 13 contains at least600 nucleotides, more preferably at least 630 nucleotides, and mostpreferably at least 660 nucleotides.

In a fourth aspect, the isolated polypeptides having cellulolyticenhancing activity are encoded by polynucleotides that hybridize underat least very low stringency conditions, preferably at least lowstringency conditions, more preferably at least medium stringencyconditions, more preferably at least medium-high stringency conditions,even more preferably at least high stringency conditions, and mostpreferably at least very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11, or the genomicDNA sequence comprising the mature polypeptide coding sequence of SEQ IDNO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, (iii) a subsequence of (i) or(ii), or (iv) a full-length complementary strand of (i), (ii), or (iii)(J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). Asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,or SEQ ID NO: 13 contains at least 100 contiguous nucleotides orpreferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment that has cellulolyticenhancing activity. In a preferred aspect, the mature polypeptide codingsequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9,nucleotides 67 to 796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQID NO: 13.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or asubsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO:12, or SEQ ID NO: 14, or a fragment thereof, may be used to design anucleic acid probe to identify and clone DNA encoding polypeptideshaving cellulolytic enhancing activity from strains of different generaor species, as described supra.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,or SEQ ID NO: 13, the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ IDNO: 11, or the genomic DNA sequence comprising the mature polypeptidecoding sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, itsfull-length complementary strand, or a subsequence thereof, under verylow to very high stringency conditions, as described supra.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 1. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pEJG120 which is containedin E. coli NRRL B-30699, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pEJG120 which is contained in E.coli NRRL B-30699.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 3. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61C which is containedin E. coli NRRL B-30813, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61C which is contained in E.coli NRRL B-30813.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, ora subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTter61D whichis contained in E. coli NRRL B-30812, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTter61 D whichis contained in E. coli NRRL B-30812.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 7. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61 E which iscontained in E. coli NRRL B-30814, wherein the polynucleotide sequencethereof encodes a polypeptide having cellulolytic enhancing activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence contained in plasmid pTter61 E which iscontained in E. coli NRRL B-30814.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9In another preferred aspect, the nucleic acid probe is a polynucleotidesequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 9. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pTter61G which is containedin E. coli NRRL B-30811, wherein the polynucleotide sequence thereofencodes a polypeptide having cellulolytic enhancing activity. In anotherpreferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence contained in plasmid pTter61G which is contained in E.coli NRRL B-30811.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 11. In another preferredaspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 11. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pDZA2-7 whichis contained in E. coli NRRL B-30704, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pDZA2-7 which iscontained in E. coli NRRL B-30704.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 13. In another preferredaspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO:13. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 13. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pTr333 whichis contained in E. coli NRRL B-30878, wherein the polynucleotidesequence thereof encodes a polypeptide having cellulolytic enhancingactivity. In another preferred aspect, the nucleic acid probe is themature polypeptide coding sequence contained in plasmid pTr333 which iscontained in E. coli NRRL B-30878.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are as defined herein.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed as defined herein.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are as defined herein.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed as defined herein.

In a fifth aspect, the polypeptides having cellulolytic enhancingactivity are encoded by polynucleotides comprising or consisting ofnucleotide sequences that have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 ofpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%,97%, 98%, or 99%, which encode an active polypeptide having cellulolyticenhancing activity.

In a preferred aspect, the mature polypeptide coding sequence isnucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ IDNO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 ofSEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 67 to796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQ ID NO: 13. Seepolynucleotide section herein.

In a sixth aspect, the polypeptides having cellulolytic enhancingactivity are artificial variants comprising a substitution, deletion,and/or insertion of one or more (or several) amino acids of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; or a homologoussequence thereof. Methods for preparing such artificial variants aredescribed supra.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14,is 10, preferably 9, more preferably 8, more preferably 7, morepreferably at most 6, more preferably 5, more preferably 4, even morepreferably 3, most preferably 2, and even most preferably 1.

Sources for Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity may be obtainedfrom microorganisms of any genus. In a preferred aspect, the polypeptideobtained from a given source is secreted extracellularly.

A polypeptide having cellulolytic enhancing activity may be a bacterialpolypeptide. For example, the polypeptide may be a gram positivebacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancingactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingcellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having cellulolytic enhancing activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingcellulolytic enhancing activity.

The polypeptide having cellulolytic enhancing activity may also be afungal polypeptide, and more preferably a yeast polypeptide such as aCandida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia polypeptide having cellulolytic enhancing activity; or morepreferably a filamentous fungal polypeptide such as aan Acremonium,Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria,Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus,Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus,Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides,Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus,Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum,Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylariapolypeptide having cellulolytic enhancing activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having cellulolyticenhancing activity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum,Penicillium purpurogenum, Phanerochaete chrysosporium, Thielaviaachromatica, Thielavia albomyces, Thielavia albopilosa, Thielaviaaustraleinsis, Thielavia fimeti, Thielavia microspora, Thielaviaovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa,Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,Trichoderma viride, or Trichophaea saccata polypeptide havingcellulolytic enhancing activity.

In a more preferred aspect, the polypeptide is a Thielavia terrestrispolypeptide having cellulolytic enhancing activity. In a most preferredembodiment, the polypeptide is a Thielavia terrestris NRRL 8126polypeptide having cellulolytic enhancing activity, e.g., the maturepolypeptide of SEQ ID NO: 2, 4, 6, 8, or 10, or fragments thereof thathave cellulolytic enhancing activity.

In another more preferred aspect, the polypeptide is a Thermoascusaurantiacus polypeptide, e.g., the mature polypeptide of SEQ ID NO: 12.

In another more preferred aspect, the polypeptide is a Trichodermareesei polypeptide having cellulolytic enhancing activity. In anothermost preferred aspect, the polypeptide is a Trichoderma reesei RutC30(ATCC 56765) polypeptide, having cellulolytic enhancing activity e.g.,the mature polypeptide of SEQ ID NO: 14, or fragments thereof that havecellulolytic enhancing activity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes, as describedherein.

Polypeptides having cellulolytic enhancing activity also include fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof having cellulolytic enhancing activity,and can further comprise a cleavage site, as described herein.

For further details on polypeptides having cellulolytic enhancingactivity and polynucleotides thereof, see WO 2005/074647, WO2005/074656, and U.S. Published Application Ser. No. 2007/0077630, whichare incorporated herein by reference.

Polynucleotides Encoding Polypeptides Having Cellulolytic EnhancingActivity

Polynucleotides comprising nucleotide sequences that encode polypeptideshaving cellulolytic enhancing activity can be isolated and utilized topractice the methods of the present invention, as described herein.

The polynucleotides comprise nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, orSEQ ID NO: 13 of preferably at least 60%, more preferably at least 65%,more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, most preferably at least 95%, and even mostpreferably at least 96%, 97%, 98%, or 99%, which encode a polypeptidehaving cellulolytic enhancing activity.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pEJG120 thatis contained in Escherichia coli NRRL B-30699. In another preferredaspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence of SEQ ID NO: 1. In another more preferredaspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pEJG120 that iscontained in Escherichia coli NRRL B-30699. The present invention alsoencompasses nucleotide sequences that encode a polypeptide having theamino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,which differ from SEQ ID NO: 1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NO: 1 that encode fragments of SEQ ID NO: 2 that have cellulolyticenhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 3. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pTter61C that is contained in Escherichia coli NRRL B-30813. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 3. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTter61Cthat is contained in Escherichia coli NRRL B-30813. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 4 or the maturepolypeptide thereof, which differ from SEQ ID NO: 3 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 3 that encode fragments of SEQ ID NO: 4 thathave cellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 5. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pTter61D that is contained in Escherichia coli NRRL B-30812. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 5. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTter61Dthat is contained in Escherichia coli NRRL B-30812. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 6 or the maturepolypeptide thereof, which differ from SEQ ID NO: 5 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 5 that encode fragments of SEQ ID NO: 6 thathave cellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 7. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pTter61 E that is contained in Escherichia coli NRRL B-30814. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 7. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTter61Ethat is contained in Escherichia coli NRRL B-30814. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 8 or the maturepolypeptide thereof, which differ from SEQ ID NO: 7 by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 7 that encode fragments of SEQ ID NO: 8 thathave cellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 9. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pTter61G that is contained in Escherichia coli NRRL B-30811. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 9. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTter61Gthat is contained in Escherichia coli NRRL B-30811. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 10 or themature polypeptide thereof, which differ from SEQ ID NO: 9 by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 9 that encode fragments of SEQ ID NO: 10that have cellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 11. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pDZA2-7 that is contained in Escherichia coli NRRL B-30704. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 11. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pDZA2-7 thatis contained in Escherichia coli NRRL B-30704. The present inventionalso encompasses nucleotide sequences that encode a polypeptide havingthe amino acid sequence of SEQ ID NO: 12 or the mature polypeptidethereof, which differ from SEQ ID NO: 11 by virtue of the degeneracy ofthe genetic code. The present invention also relates to subsequences ofSEQ ID NO: 11 that encode fragments of SEQ ID NO: 12 that havecellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 13. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pTr3337 which is contained in Escherichia coli NRRL B-30878. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 13. In anothermore preferred aspect, the nucleotide sequence comprises or consists ofthe mature polypeptide coding sequence contained in plasmid pTr3337which is contained in Escherichia coli NRRL B-30878. The presentinvention also encompasses nucleotide sequences that encode apolypeptide having the amino acid sequence of SEQ ID NO: 14 or themature polypeptide thereof, which differ from SEQ ID NO: 13 or themature polypeptide coding sequence thereof by virtue of the degeneracyof the genetic code. The present invention also relates to subsequencesof SEQ ID NO: 13 that encode fragments of SEQ ID NO: 14 that havecellulolytic enhancing activity.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, or SEQ ID NO: 13, in which the mutant nucleotide sequenceencodes the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In apreferred aspect, the mature polypeptide is amino acids 20 to 326 of SEQID NO: 2, amino acids 18 to 240 of SEQ ID NO: 4, amino acids 20 to 258of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, or amino acids20 to 304 of SEQ ID NO: 10, amino acids 23 to 250 of SEQ ID NO: 12, oramino acids 20 to 249 of SEQ ID NO: 14. In another preferred aspect, themature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ IDNO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 ofSEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, ornucleotides 77 to 766 of SEQ ID NO: 13.

As described earlier, the techniques used to isolate or clone apolynucleotide encoding a polypeptide are known in the art and includeisolation from genomic DNA, preparation from cDNA, or a combinationthereof.

The polynucleotide may also be a polynucleotide encoding a polypeptidehaving cellulolytic enhancing activity that hybridizes under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 11, or the genomic DNA sequencecomprising the mature polypeptide coding sequence of SEQ ID NO: 7, SEQID NO: 9, or SEQ ID NO: 13, or (iii) a full-length complementary strandof (i) or (ii); or allelic variants and subsequences thereof (Sambrooket al., 1989, supra), as defined herein. In a preferred aspect, themature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ IDNO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 ofSEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, ornucleotides 77 to 766 of SEQ ID NO: 13.

Nucleic Acid Constructs

An isolated polynucleotide encoding a polypeptide having cellulolyticenhancing activity or a polypeptide having cellulolytic enzyme activitymay be manipulated in a variety of ways to provide for expression of thepolypeptide by constructing a nucleic acid construct comprising anisolated polynucleotide encoding the polypeptide operably linked to oneor more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Manipulation of the polynucleotide's sequence priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesequences utilizing recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding such a polypeptide. The promoter sequencecontains transcriptional control sequences that mediate the expressionof the polypeptide. The promoter may be any nucleotide sequence thatshows transcriptional activity in the host cell of choice includingmutant, truncated, and hybrid promoters, and may be obtained from genesencoding extracellular or intracellular polypeptides either homologousor heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs, especially in a bacterial host cell, are thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding region that encodes the secreted polypeptide. Alternatively, the5′ end of the coding sequence may contain a signal peptide codingsequence that is foreign to the coding sequence. The foreign signalpeptide coding sequence may be required where the coding sequence doesnot naturally contain a signal peptide coding sequence. Alternatively,the foreign signal peptide coding sequence may simply replace thenatural signal peptide coding sequence in order to enhance secretion ofthe polypeptide. However, any signal peptide coding sequence thatdirects the expressed polypeptide into the secretory pathway of a hostcell of choice, i.e., secreted into a culture medium, may be used in thepresent invention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 19 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding region comprises or consists of nucleotides 330 to 387 ofSEQ ID NO: 1.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 17 of SEQ ID NO: 4. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 47 to97 of SEQ ID NO: 3.

In another preferred aspect, the signal peptide comprises or consists ofamino acids coding region is amino acids 1 to 19 of SEQ ID NO: 6. Inanother preferred aspect, the signal peptide coding region comprises orconsists of nucleotides 69 to 125 of SEQ ID NO: 5.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 18 of SEQ ID NO: 8. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 1 to54 of SEQ ID NO: 7.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 19 of SEQ ID NO: 10. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 1 to57 of SEQ ID NO: 9.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 22 of SEQ ID NO: 12. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 1 to66 of SEQ ID NO: 11.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 19 of SEQ ID NO: 14. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 20 to76 of SEQ ID NO: 13.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The various nucleic acids and control sequences described herein may bejoined together to produce a recombinant expression vector comprising apolynucleotide encoding a polypeptide having cellulolytic enhancingactivity or a polypeptide having cellulolytic enzyme activity, apromoter, and transcriptional and translational stop signals. Theexpression vectors may include one or more convenient restriction sitesto allow for insertion or substitution of the polynucleotide sequenceencoding the polypeptide at such sites. Alternatively, a polynucleotideencoding such a polypeptide may be expressed by inserting thepolynucleotide sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors preferably contain one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide encoding such a polypeptide maybe inserted into the host cell to increase production of thepolypeptide. An increase in the copy number of the polynucleotide can beobtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the polynucleotide where cells containing amplifiedcopies of the selectable marker gene, and thereby additional copies ofthe polynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

Host Cells

Recombinant host cells comprising a polynucleotide encoding apolypeptide having cellulolytic enhancing activity or a polypeptidehaving cellulolytic enzyme activity can be advantageously used in therecombinant production of the polypeptide. A vector comprising such apolynucleotide is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

The bacterial host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phiebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

Methods for producing a polypeptide having cellulolytic enhancingactivity or a polypeptide having cellulolytic enzyme activity, comprise(a) cultivating a cell, which in its wild-type form is capable ofproducing the polypeptide, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

Alternatively, methods for producing a polypeptide having cellulolyticenhancing activity or a polypeptide having cellulolytic enzyme activity,comprise (a) cultivating a recombinant host cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

In the production methods, the cells are cultivated in a nutrient mediumsuitable for production of the polypeptide using methods well known inthe art. For example, the cell may be cultivated by shake flaskcultivation, and small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides having cellulolytic enhancing activity or cellulolyticenzyme activity are detected using the methods described herein.

The resulting broth may be used as is or the polypeptide may berecovered using methods known in the art. For example, the polypeptidemay be recovered from the nutrient medium by conventional proceduresincluding, but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

Methods for Processing Cellulose-Containing Material

The compositions and methods of the present invention can be used tohydrolyze (saccharify) a cellulose-containing material, e.g.,lignocellulose, to fermentable sugars and convert the fermentable sugarsto many useful substances, e.g., chemicals and fuels. The production ofa desired fermentation product from cellulose-containing materialtypically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of cellulose-containing material according to the presentinvention can be accomplished using processes known in the art.Moreover, the methods of the present invention can be implemented usingany biomass processing apparatus configured to operate in accordancewith the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF), simultaneous saccharification and fermentation(SSF), simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), SHCF (separate hydrolysis andco-fermentation), HHCF (hybrid hydrolysis and fermentation), and directmicrobial conversion (DMC). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude, for example, fluidized bed, upflow blanket, immobilized, andextruder type reactors for hydrolysis and/or fermentation.

Pretreatment. In practicing the methods of the present invention, anypretreatment process known in the art can be used to disrupt the plantcell wall components of the cellulose-containing material. Thecellulose-containing material can also be subjected to pre-soaking,wetting, or conditioning prior to pretreatment using methods known inthe art. Conventional pretreatments include, but are not limited to,steam pretreatment (with or without explosion), dilute acidpretreatment, hot water pretreatment, lime pretreatment, wet oxidation,wet explosion, ammonia fiber explosion, organosolv pretreatment, andbiological pretreatment. Additional pretreatments include ultrasound,electroporation, microwave, supercritical CO₂, supercritical H₂O, andammonia percolation.

The cellulose-containing material can be pretreated before hydrolysisand/or fermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with hydrolysis, such as simultaneously with treatment ofthe cellulose-containing material with one or more cellulolytic enzymes,or other enzyme activities, to release fermentable sugars, such asglucose and/or maltose. In most cases the pretreatment step itselfresults in some conversion of biomass to fermentable sugars (even inabsence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulose-containingmaterial is heated to disrupt the plant cell wall components, includinglignin, hemicellulose, and cellulose to make the cellulose and otherfractions, e.g., hemicellulase, accessible to enzymes. The cellulosematerial is passed to or through a reaction vessel where steam isinjected to increase the temperature to the required temperature andpressure and is retained therein for the desired reaction time. Steampretreatment is preferably done at 140-230° C., more preferably at160-200° C., and most preferably at 170-190° C., where the optimaltemperature range depends on addition of a chemical catalyst. Residencetime for the steam pretreatment is preferably 1-15 minutes, morepreferably 3-12 minutes, and most preferably 4-10 minutes, where theoptimal residence time depends on the temperature range and addition ofa chemical catalyst. Steam pretreatment allows for relatively highsolids loadings, so that the cellulose-containing material is generallyonly moist during the pretreatment. The steam pretreatment is oftencombined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, AppLMicrobiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730).

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases recovery, and improves enzymatic hydrolysis (Ballesteros etal., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulose-containing material is mixedwith dilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282). Ammonia fiber explosion (AFEX) involves treatingcellulose-containing material with liquid or gaseous ammonia at moderatetemperatures such as 90-100° C. and high pressure such as 17-20 bar for5-10 minutes, where the dry matter content can be as high as 60%(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh etal., 2005, Appl. Biochem. Biotechnol. 121:1133-1141; Teymouri et al.,2005, Bioresource TechnoL 96: 2014-2018).

Organosolv pretreatment delignifies cellulose-containing material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121:219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of the hemicelluloseis removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. In one aspect, theacid concentration is in the range from preferably 0.01 to 20 wt % acid,more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulose-containing material and held at a temperature, forexample, in the range of 160-220° C., preferably 165-195° C., forperiods ranging from seconds to minutes to, e.g., 1 second to 60minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulose-containing material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulose-containing material can beunwashed or washed using any method known in the art, e.g., washed withwater.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulose-containing material. Forexample, physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulose-containingmaterial can be pretreated both physically and chemically. For instance,the pretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulose-containing material issubjected to mechanical, chemical, or physical pretreatment, or anycombination thereof, to promote the separation and/or release ofcellulose, hemicellulose and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulose-containingmaterial. Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of lignocellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known assaccharification, the pretreated cellulose-containing material ishydrolyzed to break down cellulose and alternatively also hemicelluloseto fermentable sugars, such as glucose, xylose, xylulose, arabinose,maltose, mannose, galactose, and/or soluble oligosaccharides. Thehydrolysis is performed enzymatically using a cellulolytic enzymecomposition of the present invention comprising an effective amount of apolypeptide having cellulolytic enhancing activity and solubleactivating divalent metal cation. The enzyme components of thecomposition can also be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulose-containing material(substrate) is fed gradually to, for example, an enzyme containinghydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature, and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

The optimum amounts of the enzymes and polypeptides having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of component cellulolytic proteins, the cellulosicsubstrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation).

In a preferred aspect, an effective amount of cellulolytic protein(s) tocellulose-containing material is about 0.5 to about 50 mg, preferably atabout 0.5 to about 40 mg, more preferably at about 0.5 to about 25 mg,more preferably at about 0.75 to about 20 mg, more preferably at about0.75 to about 15 mg, even more preferably at about 0.5 to about 10 mg,and most preferably at about 2.5 to about 10 mg per g ofcellulose-containing material.

In another preferred aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulose-containing material isabout 0.5 to about 50 mg, preferably at about 0.5 to about 40 mg, morepreferably at about 0.5 to about 25 mg, more preferably at about 0.75 toabout 20 mg, more preferably at about 0.75 to about 15 mg, even morepreferably at about 0.5 to about 10 mg, and most preferably at about 2.5to about 10 mg per g of cellulose-containing material.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulose-containing materialis about 0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg,more preferably about 0.01 to about 30 mg, more preferably about 0.01 toabout 20 mg, more preferably about 0.01 to about 10 mg, more preferablyabout 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5mg, more preferably at about 0.05 to about 1.25 mg, more preferably atabout 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25mg, even more preferably at about 0.15 to about 1.25 mg, and mostpreferably at about 0.25 to about 1.0 mg per g of cellulose-containingmaterial.

In another preferred aspect, an effective amount of polypeptide(s)having cellulolytic enhancing activity to cellulolytic protein(s) isabout 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g,more preferably at about 0.15 to about 0.75 g, more preferably at about0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, evenmore preferably at about 0.1 to about 0.5 g, and most preferably atabout 0.05 to about 0.2 g per g of cellulolytic protein(s).

Fermentation. The fermentable sugars obtained from the pretreated andhydrolyzed cellulose-containing material can be fermented by one or morefermenting microorganisms capable of fermenting the sugars directly orindirectly into a desired fermentation product. “Fermentation” or“fermentation process” refers to any fermentation process or any processcomprising a fermentation step. Fermentation processes also includefermentation processes used in the biofuel industry, consumable alcoholindustry (e.g., beer and wine), dairy industry (e.g., fermented dairyproducts), leather industry, and tobacco industry. The fermentationconditions depend on the desired fermentation product and fermentingorganism and can easily be determined by one skilled in the art.

In the fermentation step, sugars, released from the cellulose-containingmaterial as a result of the pretreatment and enzymatic hydrolysis steps,are fermented to a product, e.g., ethanol, by a fermenting organism,such as yeast. Hydrolysis (saccharification) and fermentation can beseparate or simultaneous. Such methods include, but are not limited to,separate hydrolysis and fermentation (SHF), simultaneoussaccharification and fermentation (SSF), simultaneous saccharificationand cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF),SHCF (separate hydrolysis and co-fermentation), HHCF (hybrid hydrolysisand fermentation), and direct microbial conversion (DMC).

Any suitable hydrolyzed cellulose-containing material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium, forexample, used in a simultaneous saccharification and fermentationprocess (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast. Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Klyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, Wis., USA), BIOFERM™ AFTand XR (available from NABC-North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca.

The fermenting microorganism(s) is typically added to the degradedcellulose or hydrolysate and the fermentation is performed for about 8to about 96 hours, such as about 24 to about 60 hours. The temperatureis typically between about 26° C. to about 60° C., in particular about32° C. or 50° C., and at about pH 3 to about pH 8, such as around pH4-5, 6, or 7.

In a preferred aspect, the fermenting microorganism(s) is applied to thedegraded cellulose or hydrolysate and the fermentation is performed forabout 12 to about 96 hours, such as typically 24-60 hours. In apreferred aspect, the temperature is preferably between about 20° C. toabout 60° C., more preferably about 25° C. to about 50° C., and mostpreferably about 32° C. to about 50° C., in particular about 32° C. or50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some, e.g., bacterial fermentingorganisms have higher fermentation temperature optima. The fermentingmicroorganism(s) is preferably applied in amounts of approximately 10⁵to 10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenoreet al., Improving ethanol production and viability of Saccharomycescerevisiae by a vitamin feeding strategy during fed-batch process,Springer-Verlag (2002), which is hereby incorporated by reference.Examples of minerals include minerals and mineral salts that can supplynutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substancederived from the fermentation. The fermentation product can be, withoutlimitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol,methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid(e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citricacid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaricacid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionicacid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); a ketone (e.g.,acetone); an aldehyde (e.g., formaldehyde); an amino acid (e.g.,aspartic acid, glutamic acid, glycine, lysine, serine, and threonine);and a gas (e.g., methane, hydrogen (H₂), carbon dioxide (CO₂), andcarbon monoxide (CO)). The fermentation product can also be protein as ahigh value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an aldehyde. Inanother more preferred aspect, the aldehyde is formaldehyde.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery. The fermentation product(s) can be optionally recovered fromthe fermentation medium using any method known in the art including, butnot limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing), differentialsolubility (e.g., ammonium sulfate precipitation), distillation, orextraction. For example, ethanol is separated from the fermentedcellulose-containing material and purified by conventional methods ofdistillation. Ethanol with a purity of up to about 96 vol.% can beobtained, which can be used as, for example, fuel ethanol, drinkingethanol, i.e., potable neutral spirits, or industrial ethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

DNA Sequencing

DNA sequencing was performed using an Applied Biosystems Model 3130XGenetic Analyzer (Applied Biosystems, Foster City, Calif., USA) usingdye terminator chemistry (Giesecke et al., 1992, Journal of Virol.Methods 38: 47-60). Sequences were assembled using phred/phrap/consed(University of Washington, Seattle, Wash., USA) with sequence specificprimers.

Media

NNCYP medium was composed per liter of 5.0 g of NH₄NO₃, 0.5 g ofMgSO₄·7H₂O, 0.3 g of CaCl₂, 2.5 g of citric acid, 1.0 g of BactoPeptone, 5.0 g of yeast extract, COVE trace metals solution, andsufficient K₂HPO₄ to achieve a final pH of approximately 5.4.

COVE trace metals solution was composed per liter of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 g of CuSO₄·5H₂O, 1.2 g of FeSO₄·7H₂O, 0.7 g of MnSO₄·H₂O, 0.8g of Na₂MoO₂·2H₂O, and 10 g of ZnSO₄·7H₂O.

YP medium was composed per liter of 10 g of yeast extract and 20 g ofbacto tryptone.

Cellulase-inducing medium was composed per liter of 20 g of cellulose,10 g of corn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 gof CaCl₂, 0.42 g of MgSO₄·7H₂O, and 0.42 ml of trace metals solution.

Trace metals solution was composed per liter of 216 g of FeCl₃·6H₂O, 58g of ZnSO₄·7H₂O, 27 g of MnSO₄·H₂O, 10 g of CuSO₄·5H₂O, 2.4 g of H₃BO₃,and 336 g of citric acid.

STC was composed of 1 M sorbitol, 10 mM CaCl₂, and 10 mM Tris-HCl, pH7.5.

COVE plates were composed per liter of 342 g of sucrose, 10 ml of COVEsalts solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl, and 25 g ofNoble agar.

COVE salts solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE2 plates were composed per liter of 30 g of sucrose, 20 ml of COVEsalts solution, 25 g of Noble agar, and 10 ml of 1 M acetamide.

PDA plates were composed per liter of 39 grams of potato dextrose agar.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, 5 g of sodium chloride.

2X YT-Amp plates were composed per liter of 10 g of tryptone, 5 g ofyeast extract, 5 g of sodium chloride, and 15 g of Bacto Agar, followedby 2 ml of a filter-sterilized solution of 50 mg/ml ampicillin afterautoclaving.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄·7H₂O, 1 g of NaCl, 2 g of K₂HSO₄, 12 g of KH₂PO₄, 2 g of urea, and500 μl of AMG trace metals solution, the pH was adjusted to 5.0 and thenfilter sterilized with a 0.22 μm filtering unit.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄·7H₂O, 2.5 g of CuSO₄·5H₂O, 0.5 g of NiCl₂·6H₂O, 13.8 g ofFeSO₄·H₂O, 8.5 g of MnSO₄·7H₂O, and 3 g of citric acid.

Minimal medium plates were composed per liter of 6 g of NaNO₃, 0.52 ofKCl, 1.52 g of KH₂PO₄, 1 ml of COVE trace metals solution, 20 g of Nobleagar, 20 ml of 50% glucose, 2.5 ml of 20% MgSO₄·7H₂O, and 20 ml ofbiotin stock solution.

Biotin stock solution was composed per liter of 0.2 g of biotin.

SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl,2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, followed by filter-sterilizedglucose to 20 mM after autoclaving.

Example 1 Construction of pMJ04 Expression Vector

Expression vector pMJ04 was constructed by PCR amplifying theTrichoderma reesei exocellobiohydrolase 1 gene (cbh1, CEL7A) terminatorfrom Trichoderma reesei RutC30 genomic DNA using primers 993429(antisense) and 993428 (sense) shown below. The antisense primer wasengineered to have a Pac I site at the 5′-end and a Spe I site at the3′-end of the sense primer.

Primer 993429 (antisense): (SEQ ID NO: 29)5′-AACGTTAATTAAGGAATCGTTTTGTGTTT-3′ Primer 993428 (sense): (SEQ ID NO:30) 5′-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3′

Trichoderma reesei RutC30 genomic DNA was isolated using a DNEASY® PlantMaxi Kit (QIAGEN Inc., Valencia, Calif., USA).

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.3 mMdNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 0.3 μM primer993429, 0.3 μM primer 993428, and 2 units of Vent DNA polymerase (NewEngland Biolabs, Beverly, Mass., USA). The reactions were incubated inan EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury,N.Y., USA) programmed for 5 cycles each for 30 seconds at 94° C., 30seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles eachfor 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72°C. (5 minute final extension). The reaction products were isolated by1.0% agarose gel electrophoresis using 40 mM Tris base-20 mM sodiumacetate-1 mM disodium EDTA (TAE) buffer where a 229 bp product band wasexcised from the gel and purified using a QIAQUICK® Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The resulting PCR fragment was digested with Pac I and Spe I and ligatedinto pAILo1 (WO 05/067531) digested with the same restriction enzymesusing a Rapid Ligation Kit (Roche, Indianapolis, Ind., USA), to generatepMJ04 (FIG. 1).

Example 2 Construction of pCaHj568

Plasmid pCaHj568 was constructed from pCaHj170 (U.S. Pat. No. 5,763,254)and pMT2188. Plasmid pCaHj170 comprises the Humicola insolensendoglucanase V (CEL45A) full-length coding region (SEQ ID NO: 31, whichencodes the amino acid sequence of SEQ ID NO: 32). Construction ofpMT2188 was initiated by PCR amplifying the pUC19 origin of replicationfrom pCaHj483 (WO 98/00529) using primers 142779 and 142780 shown below.Primer 142780 introduces a Bbu I site in the PCR fragment.

Primer 142779: (SEQ ID NO: 33) 5′-TTGAATTGAAAATAGATTGATTTAAAACTTC-3′Primer 142780: (SEQ ID NO: 34) 5′-TTGCATGCGTAATCATGGTCATAGC-3′

An EXPAND® PCR System (Roche Molecular Biochemicals, Basel, Switzerland)was used following the manufacturer's instructions for thisamplification. PCR products were separated on an agarose gel and an 1160by fragment was isolated and purified using a Jetquick Gel ExtractionSpin Kit (Genomed, Wielandstr, Germany).

The URA3 gene was amplified from the general Saccharomyces cerevisiaecloning vector pYES2 (Invitrogen, Carlsbad, Calif., USA) using primers140288 and 142778 shown below using an EXPAND® PCR System. Primer 140288introduced an Eco RI site into the PCR fragment.

Primer 140288: (SEQ ID NO: 35) 5′-TTGAATTCATGGGTAATAACTGATAT-3′ Primer142778: (SEQ ID NO: 36) 5′-AAATCAATCTATTTTCAATTCAATTCATCATT-3′

PCR products were separated on an agarose gel and an 1126 by fragmentwas isolated and purified using a Jetquick Gel Extraction Spin Kit.

The two PCR fragments were fused by mixing and amplified using primers142780 and 140288 shown above by the overlap splicing method (Horton etal., 1989, Gene 77: 61-68). PCR products were separated on an agarosegel and a 2263 by fragment was isolated and purified using a JetquickGel Extraction Spin Kit.

The resulting fragment was digested with Eco RI and Bbu I and ligatedusing standard protocols to the largest fragment of pCaHj483 digestedwith the same restriction enzymes. The ligation mixture was transformedinto pyrF-negative E. coli strain DB6507 (ATCC 35673) made competent bythe method of Mandel and Higa, 1970, J. Mol. Biol. 45: 154.Transformants were selected on solid M9 medium (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press) supplemented per liter with 1 g of casaminoacids, 500μg of thiamine, and 10 mg of kanamycin. A plasmid from one transformantwas isolated and designated pCaHj527 (FIG. 2).

The NA2-tpi promoter present on pCaHj527 was subjected to site-directedmutagenesis by PCR using an EXPAND® PCR System according to themanufacturer's instructions. Nucleotides 134-144 were converted fromGTACTAAAACC (SEQ ID NO: 37) to CCGTTAAATTT (SEQ ID NO: 38) usingmutagenic primer 141223 shown below.

Primer 141223: (SEQ ID NO: 39)5′-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCAT CCC-3′Nucleotides 423-436 were converted from ATGCAATTTAAACT (SEQ ID NO: 40)to CGGCAATTTAACGG (SEQ ID NO: 41) using mutagenic primer 141222 shownbelow.

Primer 141222: (SEQ ID NO: 42)5′-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCG-3′The resulting plasmid was designated pMT2188 (FIG. 3).

The Humicola insolens endoglucanase V coding region was transferred frompCaHj170 as a Barn HI-Sal I fragment into pMT2188 digested with Barn HIand Xho I to generate pCaHj568 (FIG. 4). Plasmid pCaHj568 comprises amutated NA2-tpi promoter operably linked to the Humicola insolensendoglucanase V full-length coding sequence.

Example 3 Construction of pMJ05

Plasmid pMJ05 was constructed by PCR amplifying the 915 by Humicolainsolens endoglucanase V full-length coding region from pCaHj568 usingprimers HiEGV-F and HiEGV-R shown below.

Primer HiEGV-F (sense): (SEQ ID NO: 43)5′-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3′ Primer HiEGV-R (antisense): (SEQ IDNO: 44) 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.3 mMdNTPs, 10 ng/μl of pCaHj568, 0.3 μM HiEGV-F primer, 0.3 μM HiEGV-Rprimer, and 2 units of Vent DNA polymerase (New England Biolabs,Beverly, Mass., USA). The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120seconds at 72° C. (5 minute final extension). The reaction products wereisolated by 1.0% agarose gel electrophoresis using TAE buffer where a937 by product band was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The 937 by purified fragment was used as template DNA for subsequentamplifications with the following primers:

Primer HiEGV-R (antisense): (SEQ ID NO: 45)5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′ Primer HiEGV-F-overlap (sense):(SEQ ID NO: 46) 5′-ACCGCGGACTGCGCATC ATGCGTTCCTCCCCCCTCC-3′Primer sequences in italics are homologous to 17 by of the promoter ofthe Trichoderma reesei cellobiohydrolase I gene (cbh1) (WO 91/17243) andunderlined primer sequences are homologous to 29 by of the Humicolainsolens endoglucanase V coding region. A 36 by overlap between thepromoter and the coding sequence allowed precise fusion of a 994 byfragment comprising the Trichoderma reesei cbh1 promoter to the 918 byfragment comprising the Humicola insolens endoglucanase V coding region.

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 937 by PCR fragment,0.3 μM HiEGV-F-overlap primer, 0.3 μM HiEGV-R primer, and 2 units ofVent DNA polymerase. The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94°C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120seconds at 72° C. (5 minute final extension). The reaction products wereisolated by 1.0% agarose gel electrophoresis using TAE buffer where a945 by product band was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

A separate PCR was performed to amplify the Trichoderma reesei cbh1promoter sequence extending from 994 by upstream of the ATG start codonof the gene from Trichoderma reesei RutC30 genomic DNA using the primersshown below (the sense primer was engineered to have a Sal I restrictionsite at the 5′-end). Trichoderma reesei RutC30 genomic DNA was isolatedusing a DNeasy Plant Maxi Kit.

Primer TrCBHIpro-F (sense): (SEQ ID NO: 47)5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′ Primer TrCBHIpro-R (antisense): (SEQID NO: 48) 5′-GATGCGCAGTCCGCGGT-3′

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng/μl Trichoderma reesei RutC30genomic DNA, 0.3 μM TrCBHIpro-F primer, 0.3 μM TrCBHlpro-R primer, and 2units of Vent DNA polymerase. The reactions were incubated in anEPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30seconds at 94° C., 30 seconds at 55° C., and 120 seconds at 72° C. (5minute final extension). The reaction products were isolated by 1.0%agarose gel electrophoresis using TAE buffer where a 998 bp product bandwas excised from the gel and purified using a QIAQUICK® Gel ExtractionKit according to the manufacturer's instructions.

The purified 998 bp PCR fragment was used as template DNA for subsequentamplifications using the primers shown below.

Primer TrCBHIpro-F: (SEQ ID NO: 49) 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′Primer TrCBHIpro-R-overlap: (SEQ ID NO: 50) 5′-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3′

Sequences in italics are homologous to 17 bp of the Trichoderma reeseicbh1 promoter and underlined sequences are homologous to 29 bp of theHumicola insolens endoglucanase V coding region. A 36 bp overlap betweenthe promoter and the coding sequence allowed precise fusion of the 994bp fragment comprising the Trichoderma reesei cbh1 promoter to the 918bp fragment comprising the Humicola insolens endoglucanase V full-lengthcoding region.

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 998 by PCR fragment,0.3 μM TrCBHlpro-F primer, 0.3 μM TrCBHlpro-R-overlap primer, and 2units of Vent DNA polymerase. The reactions were incubated in anEPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C.,followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65°C., and 120 seconds at 72° C. (5 minute final extension). The reactionproducts were isolated by 1.0% agarose gel electrophoresis using TAEbuffer where a 1017 bp product band was excised from the gel andpurified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The 1017 bp Trichoderma reesei cbh1 promoter PCR fragment and the 945 bpHumicola insolens endoglucanase V PCR fragment were used as template DNAfor subsequent amplification using the following primers to preciselyfuse the 994 bp cbh1 promoter to the 918 bp endoglucanase V full-lengthcoding region using overlapping PCR.

Primer TrCBHIpro-F: (SEQ ID NO: 51) 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′Primer HiEGV-R: (SEQ ID NO: 52) 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1X ThermoPolReaction Buffer, 0.3 mM dNTPs, 0.3 μM TrCBHlpro-F primer, 0.3 μM HiEGV-Rprimer, and 2 units of Vent DNA polymerase. The reactions were incubatedin an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C.,followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65°C., and 120 seconds at 72° C. (5 minute final extension). The reactionproducts were isolated by 1.0% agarose gel electrophoresis using TAEbuffer where a 1926 bp product band was excised from the gel andpurified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The resulting 1926 bp fragment was cloned into a pCR®-Blunt-II-TOPO®vector (Invitrogen, Carlsbad, Calif., USA) using a ZEROBLUNT® TOPO® PCRCloning Kit (Invitrogen, Carlsbad, Calif., USA) following themanufacturer's protocol. The resulting plasmid was digested with Not Iand Sal I and the 1926 bp fragment was gel purified using a QIAQUICK®Gel Extraction Kit and ligated using T4 DNA ligase (Roche, Indianapolis,Ind., USA) into pMJ04, which was also digested with the same tworestriction enzymes, to generate pMJ05 (FIG. 5). Plasmid pMJ05 comprisesthe Trichoderma reesei cellobiohydrolase I promoter and terminatoroperably linked to the Humicola insolens endoglucanase V full-lengthcoding sequence.

Example 4 Construction of pSMai130 Expression Vector

A 2586 bp DNA fragment spanning from the ATG start codon to the TAA stopcodon of an Aspergillus oryzae beta-glucosidase full-length codingsequence (SEQ ID NO: 15 for cDNA sequence and SEQ ID NO: 16 for thededuced amino acid sequence; E. coli DSM 14240) was amplified by PCRfrom pJaL660 (WO 2002/095014) as template with primers 993467 (sense)and 993456 (antisense) shown below. A Spe I site was engineered at the5′ end of the antisense primer to facilitate ligation. Primer sequencesin italics are homologous to 24 bp of the Trichoderma reesei cbh1promoter and underlined sequences are homologous to 22 bp of theAspergillus oryzae beta-glucosidase coding region.

Primer 993467: (SEQ ID NO: 53) 5′-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAGG- 3′ Primer 993456: (SEQ ID NO: 54)5′-ACTAGTTTACTGGGCCTTAGGCAGCG-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif., USA), 0.25 mM dNTPs, 10 ng ofpJaL660, 6.4 μM primer 993467, 3.2 μM primer 993456, 1 mM MgCl₂, and 2.5units of Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA). Thereactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmedfor 30 cycles each for 1 minute at 94° C., 1 minute at 55° C., and 3minutes at 72° C. (15 minute final extension). The reaction productswere isolated by 1.0% agarose gel electrophoresis using TAE buffer wherea 2586 bp product band was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

A separate PCR was performed to amplify the Trichoderma reesei cbh1promoter sequence extending from 1000 bp upstream of the ATG start codonof the gene, using primer 993453 (sense) and primer 993463 (antisense)shown below to generate a 1000 bp PCR fragment.

Primer 993453: (SEQ ID NO: 55) 5′-GTCGACTCGAAGCCCGAATGTAGGAT-3′ Primer993463: (SEQ ID NO: 56) 5′-CCTCGATCCAACCAAGCTTCATGATGCGCAGTCCGCGGTTGACTA- 3′Primer sequences in italics are homologous to 24 bp of the Trichodermareesei cbh1 promoter and underlined primer sequences are homologous to22 bp of the Aspergillus oryzae beta-glucosidase full-length codingregion. The 46 bp overlap between the promoter and the coding sequenceallowed precise fusion of the 1000 bp fragment comprising theTrichoderma reesei cbh1 promoter to the 2586 bp fragment comprising theAspergillus oryzae beta-glucosidase coding region.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA,6.4 μM primer 993453, 3.2 μM primer 993463, 1 mM MgCl₂, and 2.5 units ofPfx DNA polymerase. The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 30 cycles each for 1 minute at 94° C.,1 minute at 55° C., and 3 minutes at 72° C. (15 minute final extension).The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1000 bp product band was excised from the geland purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The purified fragments were used as template DNA for subsequentamplification by overlapping PCR using primer 993453 (sense) and primer993456 (antisense) shown above to precisely fuse the 1000 bp fragmentcomprising the Trichoderma reesei cbh1 promoter to the 2586 bp fragmentcomprising the Aspergillus oryzae beta-glucosidase full-length codingregion.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 6.4 μM primer 99353, 3.2 μM primer 993456, 1 mMMgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubatedin an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1minute at 94° C., 1 minute at 60° C., and 4 minutes at 72° C. (15 minutefinal extension).

The resulting 3586 bp fragment was digested with Sal I and Spe I andligated into pMJ04, digested with the same two restriction enzymes, togenerate pSMai130 (FIG. 6). Plasmid pSMai130 comprises the Trichodermareesei cellobiohydrolase I gene promoter and terminator operably linkedto the Aspergillus oryzae native beta-glucosidase signal sequence andcoding sequence (i.e., full-length Aspergillus oryzae beta-glucosidasecoding sequence).

Example 5 Construction of pSMai135

The Aspergillus oryzae beta-glucosidase mature coding region (minus thenative signal sequence, see FIG. 7; SEQ ID NOs: 57 and 58 for the signalpeptide and coding sequence thereof) from Lys-20 to the TAA stop codonwas PCR amplified from pJaL660 as template with primer 993728 (sense)and primer 993727 (antisense) shown below.

Primer 993728: (SEQ ID NO: 59) 5′-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3′ Primer 993727: (SEQ ID NO: 60)5′-GACTAGTCTTACTGGGCCTTAGGCAGCG-3′Sequences in italics are homologous to 20 bp of the Humicola insolensendoglucanase V signal sequence and sequences underlined are homologousto 22 bp of the Aspergillus oryzae beta-glucosidase coding region. A SpeI site was engineered into the 5′ end of the antisense primer.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl of pJaL660, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. Thereactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmedfor 30 cycles each for 1 minute at 94° C., 1 minute at 55° C., and 3minutes at 72° C. (15 minute final extension). The reaction productswere isolated by 1.0% agarose gel electrophoresis using TAE buffer wherea 2523 bp product band was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

A separate PCR amplification was performed to amplify 1000 bp of theTrichoderma reesei cbh1 promoter and 63 bp of the Humicola insolensendoglucanase V signal sequence (ATG start codon to Ala-21, FIG. 8, SEQID NOs: 61 and 62), using primer 993724 (sense) and primer 993729(antisense) shown below.

Primer 993724: (SEQ ID NO: 63) 5′-ACGCGTCGACCGAATGTAGGATTGTTATCC-3′Primer 993729: (SEQ ID NO: 64) 5′-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3′

Primer sequences in italics are homologous to 20 bp of the Humicolainsolens endoglucanase V signal sequence and underlined primer sequencesare homologous to the 22 bp of the Aspergillus oryzae beta-glucosidasecoding region.

Plasmid pMJ05, which comprises the Humicola insolens endoglucanase Vcoding region under the control of the cbh1 promoter, was used astemplate to generate a 1063 bp fragment comprising the Trichodermareesei cbh1 promoter and Humicola insolens endoglucanase V signalsequence fragment. A 42 bp of overlap was shared between the Trichodermareesei cbh1 promoter and Humicola insolens endoglucanase V signalsequence and the Aspergillus oryzae beta-glucosidase mature codingsequence to provide a perfect linkage between the promoter and the ATGstart codon of the 2523 bp Aspergillus oryzae beta-glucosidase codingregion.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl of pMJ05, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. Thereactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmedfor 30 cycles each for 1 minute at 94° C., 1 minute at 60° C., and 4minutes at 72° C. (15 minute final extension). The reaction productswere isolated by 1.0% agarose gel electrophoresis using TAE buffer wherea 1063 bp product band was excised from the gel and purified using aQIAQUICK® Gel Extraction Kit according to the manufacturer'sinstructions.

The purified overlapping fragments were used as templates foramplification using primer 993724 (sense) and primer 993727 (antisense)described above to precisely fuse the 1063 bp fragment comprising theTrichoderma reesei cbh1 promoter and Humicola insolens endoglucanase Vsignal sequence to the 2523 bp fragment comprising the Aspergillusoryzae beta-glucosidase mature coding region frame by overlapping PCR.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 6.4 μM primer 993724, 3.2 μM primer 993727, 1 mMMgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubatedin an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 1minute at 94° C., 1 minute at 60° C., and 4 minutes at 72° C. (15 minutefinal extension). The reaction products were isolated by 1.0% agarosegel electrophoresis using TAE buffer where a 3591 bp product band wasexcised from the gel and purified using a QIAQUICK® Gel Extraction Kitaccording to the manufacturer's instructions.

The resulting 3591 by fragment was digested with Sal I and Spe I andligated into pMJ04 digested with the same restriction enzymes togenerate pSMai135 (FIG. 9). Plasmid pSMai135 comprises the Trichodermareesei cellobiohydrolase I gene promoter and terminator operably linkedto the Humicola insolens endoglucanase V signal sequence and theAspergillus oryzae beta-glucosidase mature coding sequence.

Example 6 Expression of Aspergillus oryzae Beta-Glucosidase with theHumicola Insolens Endoglucanase V Secretion Signal

Plasmid pSMai135 encoding the mature Aspergillus oryzae beta-glucosidaselinked to the Humicola insolens endoglucanase V secretion signal (FIG.8) was introduced into Trichoderma reesei RutC30 by PEG-mediatedtransformation (Penttila et al., 1987, Gene 61 155-164). The plasmidcontained the Aspergillus nidulans amdS gene to enable transformants togrow on acetamide as the sole nitrogen source.

Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17hours. Mycelia was collected by filtration using a Vacuum DrivenDisposable Filtration System (Millipore, Bedford, Mass., USA) and washedtwice with deionized water and twice with 1.2 M sorbitol. Protoplastswere generated by suspending the washed mycelia in 20 ml of 1.2 Msorbitol containing 15 mg of GLUCANEX® (Novozymes A/S, Bagsværd,Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St.Louis, Mo., USA) per ml and incubating for 15-25 minutes at 34° C. withgentle shaking at 90 rpm. Protoplasts were collected by centrifuging for7 minutes at 400× g and washed twice with cold 1.2 M sorbitol. Theprotoplasts were counted using a haemacytometer and re-suspended in STCto a final concentration of 1×10⁸ protoplasts per ml. Excess protoplastswere stored in a Cryo 1° C. Freezing Container (Nalgene, Rochester,N.Y., USA) at −80° C.

Approximately 7 μg of pSMai135 digested with Pme I was added to 100 μlof protoplast solution and mixed gently, followed by 260 μl of PEGbuffer, mixed, and incubated at room temperature for 30 minutes. STC (3ml) was then added and mixed and the transformation solution was platedonto COVE plates using Aspergillus nidulans amdS selection. The plateswere incubated at 28° C. for 5-7 days. Transformants were sub-culturedonto COVE2 plates and grown at 28° C.

Sixty-seven transformants designated SMA135 obtained with pSMai135 weresubcultured onto fresh plates containing acetamide and allowed tosporulate for 7 days at 28° C.

The 67 SMA135 Trichoderma reesei transformants were cultivated in 125 mlbaffled shake flasks containing 25 ml of cellulase-inducing medium at pH6.0 inoculated with spores of the transformants and incubated at 28° C.and 200 rpm for 7 days. Trichoderma reesei RutC30 was run as a control.Culture broth samples were removed at day 7. One ml of each culturebroth was centrifuged at 15,700× g for 5 minutes in a micro-centrifugeand the supernatants transferred to new tubes. Samples were stored at 4°C. until enzyme assay. The supernatants were assayed forbeta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranoside assubstrate, as described below.

Beta-glucosidase activity was determined at ambient temperature using 25μl aliquots of culture supernatants, diluted 1:10 in 50 mM succinate pH5.0, in 200 μl of 0.5 mg/ml p-nitrophenyl-beta-D-glucopyranoside assubstrate in 50 mM succinate pH 5.0. After 15 minutes incubation thereaction was stopped by adding 100 μl of 1 M Tris-HCl pH 8.0 and theabsorbance was read spectrophotometrically at 405 nm. One unit ofbeta-glucosidase activity corresponded to production of 1 μmol ofp-nitrophenyl per minute per liter at pH 5.0, ambient temperature.Aspergillus niger beta-glucosidase (NOVOZYM™ 188, Novozymes A/S,Bagsværd, Denmark) was used as an enzyme standard.

A number of the SMA135 transformants produced beta-glucosidaseactivities several-fold higher than that of Trichoderma reesei RutC30.Transformant SMA135-04 produced the highest beta-glucosidase activity.

SDS-PAGE was carried out using CRITERION® Tris-HCl (5% resolving) gels(Bio-Rad, Hercules, Calif., USA) with the CRITERION® System (Bio-Rad,Hercules, Calif., USA). Five μl of day 7 supernatants (see above) weresuspended in 2× concentration of Laemmli Sample Buffer (Bio-Rad,Hercules, Calif., USA) and boiled in the presence of 5%beta-mercaptoethanol for 3 minutes. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, Calif., USA). Theresulting gel was stained with BIO-SAFE® Coomassie Stain (Bio-Rad,Hercules, Calif., USA).

Totally, 26 of the 38 Trichoderma reesei SMA135 transformants produced aprotein of approximately 110 kDa that was not visible in Trichodermareesei RutC30 as control. Transformant Trichoderma reesei SMA135-04produced the highest level of beta-glucosidase.

Example 7 Construction of Expression Vector pSMai140

Expression vector pSMai140 was constructed by digesting plasmidpSATe111BG41 (WO 04/099228), which carries the Aspergillus oryzaebeta-glucosidase variant BG41 full-length coding region (SEQ ID NO: 25,which encodes the amino acid sequence of SEQ ID NO: 26), with Nco I. Theresulting 1243 bp fragment was isolated by 1.0% agarose gelelectrophoresis using TAE buffer and purified using a QIAQUICK® GelExtraction Kit according to the manufacturer's instructions.

Expression vector pSMai135 was digested with Nco I and a 8286 bpfragment was isolated by 1.0% agarose gel electrophoresis using TAEbuffer and purified using a QIAQUICK® Gel Extraction Kit according tothe manufacturer's instructions. The 1243 bp Nco I digested Aspergillusoryzae beta-glucosidase variant BG41 fragment was then ligated to the8286 bp vector fragment, using T4 DNA ligase (Roche, Indianapolis, Ind.,USA) according to manufacturer's protocol, to create the expressionvector pSMai140 (FIG. 10). Plasmid pSMai140 comprises the Trichodermareesei cellobiohydrolase I(CEL7A) gene promoter and terminator operablylinked to the Humicola insolens endoglucanase V signal sequence and theAspergillus oryzae beta-glucosidase variant mature coding sequence.

Example 8 Transformation of Trichoderma reesei RutC30 with pSMai140

Plasmid pSMai140 was linearized with Pme I and transformed into theTrichoderma reesei RutC30 strain as described in Example 6. A total of100 transformants were obtained from 4 independent transformationexperiments, all of which were cultivated in shake flasks oncellulase-inducing medium, and the beta-glucosidase activity wasmeasured from the culture medium of the transformants as described inExample 6. A number of Trichoderma reesei SMA140 transformants showedbeta-glucosidase activities several fold more than that of Trichodermareesei RutC30.

The presence of the Aspergillus oryzae beta-glucosidase variant BG41protein in the culture medium was detected by SDS-polyacrylamide gelelectrophoresis as described in Example 6 and Coomassie staining fromthe same 13 culture supernatants from which enzyme activity wereanalyzed. All thirteen transformants that had high β-glucosidaseactivity, also expressed the approximately 110 KDa Aspergillus oryzaebeta-glucosidase variant BG41, at varying yields.

The highest beta-glucosidase variant expressing transformant, asevaluated by beta-glucosidase activity assay and SDS-polyacrylamide gelelectrophoresis, was designated Trichoderma reesei SMA140-43.

Example 9 Construction of Expression Vector pSaMe-F1

A DNA fragment containing 209 bp of the Trichoderma reeseicellobiohydrolase l gene promoter and the core region (nucleotides 1 to702 of SEQ ID NO: 31, which encodes amino acids 1 to 234 of SEQ ID NO:32; WO 91/17243) of the Humicola insolens endoglucanase V gene was PCRamplified using pMJ05 as template and the primers shown below.

Primer 995103: (SEQ ID NO: 65) 5′-cccaagcttagccaagaaca-3′ Primer 995137:(SEQ ID NO: 66) 5′-gggggaggaacgcatgggatctggacggc-3′

The amplification reactions (50 μl) were composed of 1× PfxAmplification Buffer, 10 mM dNTPs, 50 mM MgSO₄, 10 ng/μl of pMJ05, 50picomoles of 995103 primer, 50 picomoles of 995137 primer, and 2 unitsof Pfx DNA polymerase. The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94°C., 30 seconds at 55° C., and 60 seconds at 72° C. (3 minute finalextension).

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 911 by product band was excised from the geland purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

A DNA fragment containing 806 bp of the Aspergillus oryzaebeta-glucosidase variant BG41 gene was PCR amplified using pSMai140 astemplate and the primers shown below.

Primer 995133: (SEQ ID NO: 67) 5′-gccgtccagatccccatgcgttcctccccc-3′Primer 995111: (SEQ ID NO: 68) 5′-ccaagcttgttcagagtttc-3′

The amplification reactions (50 μl) were composed of 1× PfxAmplification Buffer, 10 mM dNTPs, 50 mM MgSO₄, 100 ng of pSMai140, 50picomoles of 995133 primer, 50 picomoles of 995111 primer, and 2 unitsof Pfx DNA polymerase. The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94°C., 30 seconds at 55° C., and 120 seconds at 72° C. (3 minute finalextension).

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 806 bp product band was excised from the geland purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The two PCR fragments above were then subjected to overlapping PCR. Thepurified overlapping fragments were used as templates for amplificationusing primer 995103 (sense) and primer 995111 (antisense) describedabove to precisely fuse the 702 bp fragment comprising 209 bp of theTrichoderma reesei cellobiohydrolase I gene promoter and the Humicolainsolens endoglucanase V core sequence to the 806 bp fragment comprisinga portion of the Aspergillus oryzae beta-glucosidase variant BG41 codingregion by overlapping PCR.

The amplification reactions (50 μl) were composed of 1× PfxAmplification Buffer, 10 mM dNTPs, 50 mM MgSO₄, 2.5 μl of each fragment(20 ng/μl), 50 picomoles of 995103 primer, 50 picomoles of 995111primer, and 2 units of high fidelity Pfx DNA polymerase. The reactionswere incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for aninitial denaturation of 3 minutes at 95° C. followed by 30 cycles eachfor 1 minute of denaturation, 1 minute annealing at 60° C., and a 3minute extension at 72° C.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing TAE buffer where a 1.7 kb product band was excised from the geland purified using a QIAQUICK® Gel Extraction Kit according to themanufacturer's instructions.

The 1.7 kb fragment was ligated into a pCR®4 Blunt vector (Invitrogen,Carlsbad, Calif., USA) according to the manufacturer's instructions. Theconstruct was then transformed into ONE SHOT® TOP10 Chemically CompetentE. coli cells (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's rapid chemical transformation procedure. Colonies wereselected and analyzed by plasmid isolation and digestion with Hind IIIto release the 1.7 kb overlapping PCR fragment.

Plasmid pSMai140 was also digested with Hind III to linearize theplasmid. Both digested fragments were combined in a ligation reactionusing a Rapid DNA Ligation Kit following the manufacturer's instructionsto produce pSaMe-F1 (FIG. 11).

E. coli XL1-Blue Subcloning-Grade Competent Cells (Stratagene, La Jolla,Calif., USA) were transformed with the ligation product. Identity of theconstruct was confirmed by DNA sequencing of the Trichoderma reeseicellobiohydrolase I gene promoter, Humicola insolens endoglucanase Vsignal sequence, Humicola insolens endoglucanase V core, Humicolainsolens endoglucanase V signal sequence, Aspergillus oryzaebeta-glucosidase variant BG41, and the Trichoderma reeseicellobiohydrolase I gene terminator sequence from plasmids purified fromtransformed E. coli. One clone containing the recombinant plasmid wasdesignated pSaMe-F1. Plasmid pSaMe-F1 comprises the Trichoderma reeseicellobiohydrolase I gene promoter and terminator and the Humicolainsolens endoglucanase V signal peptide sequence linked directly to theHumicola insolens endoglucanase V core polypeptide, which are fuseddirectly to the Humicola insolens endoglucanase V signal peptide, whichis linked directly to the Aspergillus oryzae beta-glucosidase variantBG41 mature coding sequence. The DNA sequence and deduced amino acidsequence of the Aspergillus oryzae beta-glucosidase variant BG fusionprotein is shown in FIGS. 12A, 12B, 12C, and 12D (SEQ ID NOs: 25 and 26,respectively).

Example 10 Transformation of Trichoderma reesei RutC30 with pSaMe-F1

Shake flasks containing 25 ml of YP medium supplemented with 2% glucoseand 10 mM uridine were inoculated with 5×10⁷ spores of Trichodermareesei RutC30. Following incubation overnight for approximately 16 hoursat 27° C., 90 rpm, the mycelia were collected using a Vacuum DrivenDisposable Filtration System. The mycelia were washed twice in 100 ml ofdeionized water and twice in 1.2 M sorbitol. Protoplasts were generatedas described in Example 6.

Two micrograms of pSaMe-F1 DNA linearized with Pme I, 100 μl ofTrichoderma reesei RutC30 protoplasts, and 50% PEG (4000) were mixed andincubated for 30 minutes at room temperature. Then 3 ml of STC wereadded and the contents were poured onto a COVE plate supplemented with10 mM uridine. The plate was then incubated at 28° C. Transformantsbegan to appear by day 6 and were picked to COVE2 plates for growth at28° C. and 6 days. Twenty-two Trichoderma reesei transformants wererecovered.

Transformants were cultivated in shake flasks on cellulase-inducingmedium, and beta-glucosidase activity was measured as described inExample 6. A number of pSaMe-F1 transformants produced beta-glucosidaseactivity. One transformant, designated Trichoderma reesei SaMeF1-9,produced the highest amount of beta-glucosidase, and had twice theactivity of a strain expressing the Aspergillus oryzae beta-glucosidasevariant (Example 9).

Endoglucanase activity was assayed using a carboxymethyl cellulose (CMC)overlay assay according to Beguin, 1983, Analytical Biochem. 131(2):333-336. Five μg of total protein from five of the broth samples (thosehaving the highest beta-glucosidase activity) were diluted in NativeSample Buffer (Bio-Rad, Hercules, Calif., USA) and run on a CRITERION®8-16% Tris-HCl gel (Bio-Rad, Hercules, Calif., USA) using 10×Tris/glycine running buffer (Bio-Rad, Hercules, Calif., USA) and thenthe gel was laid on top of a plate containing 1% carboxymethylcellulose(CMC). After 1 hour incubation at 37° C., the gel was stained with 0.1%Congo Red for 20 minutes. The plate was then destained using 1 M NaCl inorder to identify regions of clearing indicative of endoglucanaseactivity. Two clearing zones were visible, one upper zone around 110 kDaand a lower zone around 25 kDa. The predicted protein size of theHumicola insolens endoglucanase V and Aspergillus oryzaebeta-glucosidase variant BG41 fusion is 118 kDa if the two proteins arenot cleaved and remain as a single polypeptide; glycosylation of theindividual endoglucanase V core domain and of the beta-glucosidase leadsto migration of the individual proteins at higher mw than predicted fromthe primary sequence. If the two proteins are cleaved then the predictedsizes for the Humicola insolens endoglucanase V core domain is 24 kDaand 94 kDa for Aspergillus oryzae beta-glucosidase variant BG41. Sincethere was a clearing zone at 110 kDa this result indicated thatminimally a population of the endoglucanase and beta-glucosidase fusionprotein remains intact as a single large protein. The lower clearingzone most likely represents the endogenous endoglucanase activity, andpossibly additionally results from partial cleavage of the Humicolainsolens endoglucanase V core domain from the Aspergillus oryzaebeta-glucosidase.

The results demonstrated the Humicola insolens endoglucanase V core wasactive even though it was linked to the Aspergillus oryzaebeta-glucosidase. In addition, the increase in beta-glucosidase activityappeared to result from increased secretion of protein relative to thesecretion efficiency of the non-fusion beta-glucosidase. By linking theAspergillus oryzae beta-glucosidase variant BG41 sequence to theefficiently secreted Humicola insolens endoglucanase V core, morebeta-glucosidase was secreted.

Example 11 Construction of Vector pSaMe-FX

Plasmid pSaMe-FX was constructed by modifying pSaMe-F1. Plasmid pSaMe-F1was digested with Bst Z17 and Eco RI to generate a 1 kb fragment thatcontained the beta-glucosidase variant BG41 coding sequence and a 9.2 kbfragment containing the remainder of the plasmid. The fragments wereseparated by 1.0% agarose gel electrophoresis using TAE buffer and the9.2 kb fragment was excised and purified using a QIAQUICK® GelExtraction Kit according to the manufacturer's instructions. PlasmidpSMai135 was also digested with Bst Z17 and Eco RI to generate a 1 kbfragment containing bases homologous to the Aspergillus oryzaebeta-glucosidase variant BG41 coding sequence and a 8.5 kb fragmentcontaining the remainder of the plasmid. The 1 kb fragment was isolatedand purified as above.

The 9.2 kb and 1 kb fragments were combined in a ligation reaction usinga Rapid DNA Ligation Kit following the manufacturer's instructions toproduce pSaMe-FX, which is identical to pSaMe-F1 except that itcontained the wild-type beta-glucosidase mature coding sequence ratherthan the variant mature coding sequence.

E. coli SURE® Competent Cells (Stratagene, La Jolla, Calif., USA) weretransformed with the ligation product. Identity of the construct wasconfirmed by DNA sequencing of plasmids purified from transformed E.coli demonstrating the presence of the Trichoderma reeseicellobiohydrolase I gene promoter, Humicola insolens endoglucanase Vsignal sequence, Humicola insolens endoglucanase V core sequence,Humicola insolens endoglucanase V signal sequence, Aspergillus oryzaebeta-glucosidase mature coding sequence, and the Trichoderma reeseicellobiohydrolase I gene terminator sequence One clone containing therecombinant plasmid was designated pSaMe-FX (FIG. 13). The DNA sequenceand deduced amino acid sequence of the Aspergillus oryzaebeta-glucosidase fusion protein is shown in FIGS. 14A, 14B, 14C, and 14D(SEQ ID NOs: 27 and 28, respectively).

Example 12 Transformation and Expression of Trichoderma Transformants

The pSaMe-FX construct was linearized with Pme I and transformed intothe Trichoderma reesei RutC30 strain as described in Example 10. A totalof 63 transformants were obtained from a single transformation.Transformants were cultivated in shake flasks on cellulase-inducingmedium, and beta-glucosidase activity was measured as described inExample 6. A number of pSaMe-FX transformants produced beta-glucosidaseactivity. One transformant designated SaMe-FX16 produced twice theamount of beta-glucosidase activity compared to Trichoderma reeseiSaMeF1-9 (Example 10).

Example 13 Analysis of Trichoderma Reesei Transformants

A fusion protein was constructed as described in Example 9 by fusing theHumicola insolens endoglucanase V core (containing its own native signalsequence) with the Aspergillus oryzae beta-glucosidase variant BG41mature coding sequence linked to the Humicola insolens endoglucanase Vsignal sequence. This fusion construct resulted in a two-fold increasein secreted beta-glucosidase activity compared to the Aspergillus oryzaebeta-glucosidase variant BG41 mature coding sequence linked to theHumicola insolens endoglucanase V signal sequence. A second fusionconstruct was made as described in Example 11 consisting of the Humicolainsolens endoglucanase V core (containing its own signal sequence) fusedwith the Aspergillus oryzae wild-type beta-glucosidase coding sequencelinked to the Humicola insolens endoglucanase V signal sequence, andthis led to an even further improvement in beta-glucosidase activity.The strain transformed with the wild-type fusion had twice the secretedbeta-glucosidase activity relative to the strain transformed with thebeta-glucosidase variant BG41 fusion.

Example 14 Cloning of the Beta-Glucosidase Fusion Protein EncodingSequence into an Aspergillus Oryzae Expression Vector

Two synthetic oligonucleotide primers, shown below, were designed to PCRamplify the full-length open reading frame from pSaMeFX encoding thebeta-glucosidase fusion protein.

PCR Forward primer: (SEQ ID NO: 69) 5′-GGACTGCGCAGCATGCGTTC-3′ PCRReverse primer: (SEQ ID NO: 70) 5′-AGTTAATTAATTACTGGGCCTTAGGCAGCG-3′Bold letters represent coding sequence. The underlined “G” in theforward primer represents a base change introduced to create an Sph Irestriction site. The remaining sequence contains sequence identitycompared with the insertion sites of pSaMeFX. The underlined sequence inthe reverse primer represents a Pac I restriction site added tofacilitate cloning into the expression vector pAlLo2 (WO 04/099228).

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 50 ng of pSaMeFX DNA, 1× Pfx Amplification Buffer, 6 μl of 10mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNAPolymerase, and 1 μl of 50 mM MgSO₄ in a final volume of 50 μl AnEPPENDORF® MASTERCYCLER® 5333 was used to amplify the fragmentprogrammed for 1 cycle at 98° C. for 2 minutes; and 35 cycles each at96° C. for 30 seconds, 61° C. for 30 seconds, and 68° C. for 3 minutes.After the 35 cycles, the reaction was incubated at 68° C. for 10 minutesand then cooled at 10° C. until further processed. A 3.3 kb PCR reactionproduct was isolated on a 0.8% GTG-agarose gel (Cambrex Bioproducts OneMeadowlands Plaza East Rutherford, N.J., USA) using TAE buffer and 0.1μg of ethidium bromide per ml. The DNA was visualized with the aid of aDARK READER™ (Clare Chemical Research, Dolores, Colo., USA) to avoidUV-induced mutations. A 3.3 kb DNA band was excised with a disposablerazor blade and purified with an ULTRAFREE®-DA spin cup (Millipore,Billerica, Mass., USA) according to the manufacturer's instructions.

The purified 3.3 kb PCR product was cloned into a pCR®4Blunt-TOPO®vector (Invitrogen, Carlsbad, Calif., USA). Four microliters of thepurified PCR product were mixed with 1 μl of a 2 M sodium chloridesolution and 1 μl of the TOPO® vector. The reaction was incubated atroom temperature for 15 minutes and then 2 μl of the reaction were usedto transform One Shot® TOP10 Chemically Competent E. coli cellsaccording to the manufacturer's instructions. Three aliquots of 83 μleach of the transformation reaction were spread onto three 150 mm 2× YTplates supplemented with 100 μg of ampicillin per ml and incubatedovernight at 37° C.

Eight recombinant colonies were used to inoculate liquid culturescontaining 3 ml of LB medium supplemented with 100 μg of ampicillin perml. Plasmid DNA was prepared from these cultures using a BIOROBOT® 9600(QIAGEN Inc., Valencia, Calif., USA). Clones were analyzed byrestriction enzyme digestion with Pac I. Plasmid DNA from each clone wasdigested with Pac I and analyzed by 1.0% agarose gel electrophoresisusing TAE buffer. All eight clones had the expected restriction digestpattern and clones 5, 6, 7, and 8 were selected to be sequenced toconfirm that there were no mutations in the cloned insert. Sequenceanalysis of their 5′ and 3′ ends indicated that all 4 clones had thecorrect sequence. Clones 5 and 7 were selected for further sequencing.Both clones were sequenced to Phred Q values of greater than 40 toensure that there were no PCR induced errors. Clones 5 and 7 were shownto have the expected sequence and clone 5 was selected for re-cloninginto pAlLo2.

Plasmid DNA from clone 5 was linearized by digestion with Sph I. Thelinearized clone was then blunt-ended by adding 1.2 μl of a 10 mM blendof dATP, dTTP, dGTP, and dCTP and 6 units of T4 DNA polymerase (NewEngland Bioloabs, Inc., Ipswich, Mass., USA). The mixture was incubatedat 12° C. for 20 minutes and then the reaction was stopped by adding 1μl of 0.5 M EDTA and heating at 75° C. for 20 minutes to inactivate theenzyme. A 3.3 kb fragment encoding the beta-glucosidase fusion proteinwas purified by gel electrophoresis and ultrafiltration as describedabove.

The vector pAlLo2 was linearized by digestion with Nco I. The linearizedvector was then blunt-ended by adding 0.5 μl of a 10 mM blend of dATP,dTTP, dGTP, and dCTP and one unit of DNA polymerase I. The mixture wasincubated at 25° C. for 15 minutes and then the reaction was stopped byadding 1 μl of 0.5 M EDTA and heating at 75° C. for 15 minutes toinactivate the enzyme. Then the vector was digested with Pac I. Theblunt-ended vector was purified by gel electrophoresis andultrafiltration as described above.

Cloning of the 3.3 kb fragment encoding the beta-glucosidase fusionprotein into the linearized and purified pAlLo2 vector was performedwith a Rapid Ligation Kit. A 1 μl sample of the reaction was used totransform E. coli XL10 SOLOPACK® Gold cells (Stratagene, La Jolla,Calif., USA) according to the manufacturer's instructions. After therecovery period, two 100 μl aliquots from the transformation reactionwere plated onto two 150 mm 2× YT plates supplemented with 100 μg ofampicillin per ml and incubated overnight at 37° C. A set of eightputative recombinant clones was selected at random from the selectionplates and plasmid DNA was prepared from each one using a BIOROBOT®9600. Clones 1-4 were selected for sequencing with pAlLo2-specificprimers to confirm that the junction vector/insert had the correctsequence. Clone 3 had a perfect vector/insert junction and wasdesignated pAlLo47 (FIG. 15).

In order to create a marker-free expression strain, a restrictionendonuclease digestion was performed to separate the blaA gene thatconfers resistance to the antibiotic ampicillin from the rest of theexpression construct. Thirty micrograms of pAlLo47 were digested withPme I. The digested DNA was then purified by agarose gel electrophoresisas described above. A 6.4 kb DNA band containing the expressionconstruct but lacking the blaA gene was excised with a razor blade andpurified with a QIAQUICK® Gel Extraction Kit.

Example 15 Expression of the Humicola Insolens/Aspergillus Oryzaecel45A-cel3A Fusion Gene in Aspergillus Oryzae JaL355

Aspergillus oryzae JaL355 (WO 00/240694) protoplasts were preparedaccording to the method of Christensen et al., 1988, Bio/Technology 6:1419-1422. Ten microliters of the purified expression construct ofExample 14 were used to transform Aspergillus oryzae JaL355 protoplasts.The transformation of Aspergillus oryzae JaL355 yielded approximately 90transformants. Fifty transformants were isolated to individual PDAplates and incubated for five days at 34° C.

Forty-eight confluent spore plates were washed with 3 ml of 0.01% TWEEN®80 and the spore suspension was used to inoculate 25 ml of MDU2BP mediumin 125 ml glass shake flasks. Transformant cultures were incubated at34° C. with constant shaking at 200 rpm. After 5 days, 1 ml aliquots ofeach culture was centrifuged at 12,000× g and their supernatantscollected. Five μl of each supernatant were mixed with an equal volumeof 2× loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm8%-16% Tris-Glycine SDS-PAGE gel and stained with BIO-SAFE® CoomassieBlue G250 protein stain (Bio-Rad, Hercules, Calif., USA). SDS-PAGEprofiles of the culture broths showed that 33 out of 48 transformantswere capable of expressing a new protein with an apparent molecularweight very close to the expected 118 kDa. Transformant 21 produced thebest yield and was selected for further studies.

Example 16 Single Spore Isolation of Aspergillus Oryzae JaL355Transformant 21

Aspergillus oryzae JaL355 transformant 21 spores were spread onto a PDAplate and incubated for five days at 34° C. A small area of theconfluent spore plate was washed with 0.5 ml of 0.01% TWEEN® 80 toresuspend the spores. A 100 μl aliquot of the spore suspension wasdiluted to a final volume of 5 ml with 0.01% TWEEN® 80. With the aid ofa hemocytometer the spore concentration was determined and diluted to afinal concentration of 0.1 spores per microliter. A 200 μl aliquot ofthe spore dilution was spread onto 150 mm Minimal medium plates andincubated for 2-3 days at 34° C. Emerging colonies were excised from theplates and transferred to PDA plates and incubated for 3 days at 34° C.Then the spores were spread across the plates and incubated again for 5days at 34° C.

The confluent spore plates were washed with 3 ml of 0.01% TWEEN® 80 andthe spore suspension was used to inoculate 25 ml of MDU2BP medium in 125ml glass shake flasks. Single-spore cultures were incubated at 34° C.with constant shaking at 200 rpm. After 5 days, a 1 ml aliquot of eachculture was centrifuged at 12,000× g and their supernatants collected.Five μl of each supernatant were mixed with an equal volume of 2×loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm8%-16% Tris-Glycine SDS-PAGE gel and stained with BIO-SAFE® CommassieBlue G250 protein stain. SDS-PAGE profiles of the culture broths showedthat all eight transformants were capable of expressing thebeta-glucosidase fusion protein at very high levels and one of culturesdesignated Aspergillus oryzae JaL355AILo47 produced the best yield.

Example 17 Construction of pCW087

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Thermoascus aurantiacus GH61A polypeptide gene from plasmidpDZA2-7 (WO 2005/074656). The forward primer results in a blunt 5′ endand the reverse primer incorporates a Pac I site at the 3′ end.

Forward Primer: (SEQ ID NO: 71) 5′-ATGTCCTTTTCCAAGATAATTGCTACTG-3′Reverse Primer: (SEQ ID NO: 72) 5′-GCTTAATTAACCAGTATACAGAGGAG-3′

Fifty picomoles of each of the primers above were used in a PCR reactionconsisting of 50 ng of pDZA2-7, 1 μl of 10 mM blend of dATP, dTTP, dGTP,and dCTP, 5 μl of 10×ACCUTAQ™ DNA Polymerase Buffer (Sigma-Aldrich, St.Louis, Mo., USA), and 5 units of ACCUTAQ™ DNA Polymerase (Sigma-Aldrich,St. Louis, Mo., USA), in a final volume of 50 μl. An EPPENDORF®MASTERCYCLER® 5333 was used to amplify the DNA fragment programmed for 1cycle at 95° C. for 3 minutes; 30 cycles each at 94° C. for 45 seconds,55° C. for 60 seconds, and 72° C. for 1 minute 30 seconds. After the 25cycles, the reaction was incubated at 72° C. for 10 minutes and thencooled at 4° C. until further processing. The 3′ end of the Thermoascusaurantiacus GH61A PCR fragment was digested using Pac I. The digestionproduct was purified using a MINELUTE™ Reaction Cleanup Kit (QIAGENInc., Valencia, Calif., USA) according to the manufacturer'sinstructions.

The GH61A fragment was directly cloned into pSMai155 (WO 2005/074647)utilizing a blunted Nco I site at the 5′ end and a Pac I site at the 3′end. Plasmid pSMai155 was digested with Nco I and Pac I. The Nco I sitewas then rendered blunt using Klenow enzymes to fill in the 5′ recessedNco I site. The Klenow reaction consisted of 20 μl of the pSma155digestion reaction mix plus 1 mM dNTPs and 1 μl of Klenow enzyme, whichwas incubated briefly at room temperature. The newly linearized pSMai155plasmid was purified using a MINELUTE™ Reaction Cleanup Kit according tothe manufacturer's instructions. These reactions resulted in thecreation a 5′ blunt end and 3′ Pac I site compatible to the newlygenerated GH61A fragment. The GH61A fragment was then cloned intopSMai155 expression vector using a Rapid DNA Ligation Kit (Roche,Indianapolis, Ind., USA) following the manufacturer's instructions. E.coli XL1-Blue Subcloning-Grade Competent Cells (Stratagene, La Jolla,Calif., USA) were transformed with the ligation product. Identity of theconstruct was confirmed by DNA sequencing of the GH61A coding sequencefrom plasmids purified from transformed E. coli. One E. coli clonecontaining the recombinant plasmid was designated pCW087-8.

Example 18 Construction of pSaMe-Ta61A

Expression vector pSaMe-Ta61 was constructed by digesting plasmid pMJ09(WO 2005/056772), which harbors the amdS selectable marker, with Nsi I,which liberated a 2.7 kb amdS fragment. The 2.7 kb amdS fragment wasthen isolated by 1.0% agarose gel electrophoresis using TAE buffer andpurified using a QIAQUICK® Gel Extraction Kit.

Expression vector pCW087 was digested with Nsi I and a 4.7 kb fragmentwas isolated by 1.0% agarose gel electrophoresis using TAE buffer andpurified using a QIAQUICK® Gel Extraction Kit. The 2.7 kb amdS fragmentwas then ligated to the 4.7 kb vector fragment, using T4 DNA ligase(Roche, Indianapolis, Ind., USA) according to manufacturer's protocol,to create the expression vector pSaMe-Ta61A. Plasmid pSaMe-Ta61Acomprises the Trichoderma reesei cellobiohydrolase I (CEL7A) genepromoter and terminator operably linked to the Thermoascus aurantiacusGH61A mature coding sequence.

Example 19 Construction of Trichoderma Reesei Strain SaMe-MF268

A co-transformation was utilized to introduce plasmids pSaMe-FX andpSaMe-Ta61A into Trichoderma reesei RutC30. Plasmids pSaMe-FX andpSaMe-Ta61A were introduced into Trichoderma reesei RutC30 byPEG-mediated transformation (Penttila et al., 1987, supra). Each plasmidcontained the Aspergillus nidulans amdS gene to enable transformants togrow on acetamide as the sole nitrogen source.

Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17hours. Mycelia were collected by filtration using a Vacuum DrivenDisposable Filtration System and washed twice with deionized water andtwice with 1.2 M sorbitol. Protoplasts were generated by suspending thewashed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX®(Novozymes A/S, Bagsværd, Denmark) per ml and 0.36 units of chitinase(Sigma Chemical Co., St. Louis, Mo., USA) per ml and incubating for15-25 minutes at 34° C. with gentle shaking at 90 rpm. Protoplasts werecollected by centrifuging for 7 minutes at 400× g and washed twice withcold 1.2 M sorbitol. The protoplasts were counted using a haemacytometerand re-suspended in STC to a final concentration of 1×10⁸ protoplastsper ml. Excess protoplasts were stored in a Cryo 1° C. FreezingContainer (Nalgene, Rochester, N.Y., USA) at −80° C.

Approximately 4 μg each of plasmids pSaMe-FX and pSaMe-Ta61A weredigested with Pme I to facilitate removal of the antibiotic resistancemarker, ampR. Following digestion with Pme I the linear fragments wererun on a 1% agarose gel using TAE buffer to separate the variousfragments. A 7.5 kb fragment from pSaMe-FX and a 4.7 kb fragment frompSaMe-Ta61A were cut out of the gel and purified using a QIAQUICK® GelExtraction Kit according to the manufacturer's instructions. Thesepurified fragments contain the amdS selectable marker cassette, theTrichoderma reesei cbh1 gene promoter and terminator; additionally, thefragment includes the Humicola insolens EGV core/Aspergillus oryzae BGfusion coding sequence or the T. aurantiacus GH61A coding sequence. Thefragments used in transformation did not contain antibiotic resistancemarkers, as the ampR fragment was removed by this gel purification step.The purified fragments were then added to 100 μl of protoplast solutionand mixed gently, followed by 260 μl of PEG buffer, mixed, and incubatedat room temperature for 30 minutes. STC (3 ml) was then added and mixedand the transformation solution was plated onto COVE plates using amdSselection. The plates were incubated at 28° C. for 5-7 days.Transformants were sub-cultured onto COVE2 plates and grown at 28° C.

Over 400 transformants were subcultured onto fresh plates containingacetamide and allowed to sporulate for 7 days at 28° C.

The Trichoderma reesei transformants were cultivated in 125 ml baffledshake flasks containing 25 ml of cellulase-inducing medium at pH 6.0inoculated with spores of the transformants and incubated at 28° C. and200 rpm for 5 days. Trichoderma reesei RutC30 was run as a control.Culture broth samples were removed at day 5. One ml of each culturebroth was centrifuged at 15,700× g for 5 minutes in a micro-centrifugeand the supernatants transferred to new tubes.

SDS-PAGE was carried out using CRITERION® Tris-HCl (5% resolving) gelswith the CRITERION® System. Five μl of day 5 supernatants (see above)were suspended in 2× concentration of Laemmli Sample Buffer (Bio-Rad,Hercules, Calif., USA) and boiled in the presence of 5%beta-mercaptoethanol for 3 minutes. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, Calif., USA). Theresulting gel was stained with BIO-SAFE® Coomassie Stain. Transformantsshowing expression of both the Thermoascus aurantiacus GH61A polypeptideand the fusion protein consisting of the Humicola insolens endoglucanaseV core (CeI45A) fused with the Aspergillus oryzae beta-glucosidase asseen by visualization of bands on SDS-PAGE gels were then tested in PCShydrolysis reactions to identify the strains producing the besthydrolytic broths.

Example 20 Identification of Trichoderma Reesei Strain SaMe-MF268

The transformants showing expression of both the Thermoascus aurantiacusGH61A polypeptide and the Aspergillus oryzae beta-glucosidase fusionprotein were cultivated in 125 ml baffled shake flasks containing 25 mlof cellulase-inducing medium at pH 6.0 inoculated with spores of thetransformants and incubated at 28° C. and 200 rpm for 5 days.

The shake flask culture broths were centrifuged at 6000× g and filteredusing STERICUP™ EXPRESS™ (Millipore, Bedford, Mass., USA) to 0.22 μmprior to hydrolysis. The activity of the culture broths was measured bytheir ability to hydrolyze the PCS and produce sugars detectable by achemical assay of their reducing ends.

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL), Boulder, Colo., USA, using dilutesulfuric acid. The following conditions were used for the pretreatment:0.048 g sulfuric acid/g dry biomass at 190° C. and 25% w/w dry solidsfor around 1 minute. The water-insoluble solids in the pretreated cornstover (PCS) contained 59.2% cellulose as determined by a limit digestof PCS to release glucose and cellobiose. Prior to enzymatic hydrolysis,the PCS was washed with a large volume of double deionized water; thedry weight of the water-washed PCS was found to be 17.73%.

PCS in the amount of 1 kg was suspended in approximately 20 liters ofdouble deionized water and, after the PCS settled, the water wasdecanted. This was repeated until the wash water was above pH 4.0, atwhich time the reducing sugars were lower than 0.06 g per liter. Forsmall volume assays (e.g., 1 ml) the settled slurry was sieved through100 Mesh screens to ensure ability to pipette. Percent dry weightcontent of the washed PCS was determined by drying the sample at a 105°C. oven for at least 24 hours (until constant weight) and comparing tothe wet weight.

PCS hydrolysis was performed in a 1 ml volume in 96-deep-well plates(Axygen Scientific) heat sealed by an ALPS 300™ automated lab platesealer (ABgene Inc., Rochester, N.Y., USA). PCS concentration was 10 gper liter in 50 mM sodium acetate pH 5.0. PCS hydrolysis was performedat 50° C. without additional stirring except during sampling asdescribed. Each reaction was performed in triplicate. Released reducingsugars were analyzed by p-hydroxy benzoic acid hydrazide (PHBAH) reagentas described below.

A volume of 0.8 ml of PCS (12.5 g per liter in water) was pipetted intoeach well of 96-deep-well plates, followed by 0.10 ml of 0.5 M sodiumacetate pH 5.0, and then 0.10 ml of diluted enzyme solution to start thereaction with a final reaction volume of 1.0 ml and PCS concentration of10 g per liter. Plates were sealed. The reaction mixture was mixed byinverting the deep-well plate at the beginning of hydrolysis and beforetaking each sample time point. At each sample time point the plate wasmixed and then the deep-well plate was centrifuged (SORVALL® RT7 withRTH-250 rotor) at 2000 rpm for 10 minutes before 20 μl of hydrolysate(supernatant) was removed and added to 180 μl of 0.4% NaOH in a 96-wellmicroplate. This stopped solution was further diluted into the properrange of reducing sugars, when necessary. The reducing sugars releasedwere assayed by para-hydroxy benzoic acid hydrazide reagent (PHBAH,Sigma, 4-hydroxy benzyhydrazide): 50 μl of PHBAH reagent (1.5%) weremixed with 100 μl of sample in a V-bottom 96-well THERMOWELL™ plate(Costar 6511), incubated on a plate heating block at 95° C. for 10minutes, and then 50 μl of double deionized water was added to eachwell, mixed, and 100 μl was transferred to another flat-bottom 96-wellplate (Costar 9017) and absorbance read at 410 nm. Reducing sugar wascalculated using a glucose calibration curve under the same conditions.Percent conversion of cellulose to reducing sugars was calculated as:

% conversion=reducing sugars (mg/ml)/(cellulose added (mg/ml)×1.11)

The factor 1.11 corrects for the weight gain in hydrolyzing cellulose toglucose.

Following the 1 ml PCS hydrolysis testing, the top candidates were grownin duplicate in fermentors according to the following protocol. Onehundred ml of the following shake flask medium was added to a 500 mlshake flask. The shake flask medium was composed per liter of 20 g ofdextrose, 10 g of corn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g ofKH₂PO₄, 0.36 g of CaCl₂, 0.42 g of MgSO₄·7H₂O, and 0.42 ml of tracemetals solution. Trace metals solution was composed per liter of 216 gof FeCl₃·6H₂O, 58 g of ZnSO₄·7H₂O, 27 g of MnSO₄·H₂O, 10 g ofCuSO₄·5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid. The shake flaskwas inoculated with two plugs from a solid plate culture of Trichodermareesei SMA135-04 and incubated at 28° C. on an orbital shaker at 200 rpmfor 48 hours. Fifty ml of the shake flask broth was used to inoculate a3 liter fermentation vessel containing 1.8 liters of a fermentationbatch medium composed per liter of 30 g of cellulose, 4 g of dextrose,10 g of corn steep solids, 3.8 g of (NH₄)₂SO₄, 2.8 g of KH₂PO₄, 2.64 gof CaCl₂, 1.63 g of MgSO₄.7H₂O, 1.8 ml of anti-foam, and 0.66 ml oftrace metals solution. Trace metals solution was composed per liter of216 g of FeCl₃·6H₂O, 58 g of ZnSO₄·7H₂O, 27 g of MnSO₄·H₂O, 10 g ofCuSO₄·5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid. Fermentation feedmedium was composed of dextrose and cellulose, which was dosed at a rateof 0 to 4 g/l/hr for a period of 165 hours. The fermentation vessel wasmaintained at a temperature of 28° C. and pH was controlled to aset-point of 4.75+/−0.1. Air was added to the vessel at a rate of 1 vvmand the broth was agitated by Rushton impeller rotating at 1100 to 1300rpm.

Total protein concentration was determined and broths were re-tested in50 g PCS hydrolysis reactions as described below. Enzyme dilutions wereprepared fresh before each experiment from stock enzyme solutions, whichwere stored at 4° C.

Hydrolysis of PCS was conducted using 125 ml screw-top Erlenmeyer flasks(VWR, West Chester, Pa., USA) using a total reaction mass of 50 gaccording to NREL Laboratory Analytical Protocol #008. In this protocolhydrolysis of PCS (approximately 11.4% in PCS and 6.8% cellulose inaqueous 50 mM sodium acetate pH 5.0 buffer) was performed usingdifferent protein loadings (expressed as mg of protein per gram ofcellulose) of the 2 liter fermentation broth samples, described above.Testing of PCS hydrolyzing capability was performed at 50° C. withorbital shaking at 150 rpm using an INNOVA® 4080 Incubator (NewBrunswick Scientific, Edison, N.J., USA). Aliquots were taken during thecourse of hydrolysis at 72, 120, and 168 hours and centrifuged, and thesupernatant liquid was filtered using a MULTISCREEN® HV 0.45 μm membrane(Millipore, Billerica, Mass., USA) by centrifugation at 2000 rpm for 10minutes using a SORVALL® RT7 plate centrifuge (Thermo Fisher Scientific,Waltham, Mass., USA). When not used immediately, filtered sugaryaliquots were frozen at −20° C. Sugar concentrations of samples dilutedin 0.005 M H₂SO₄ were measured after elution by 0.005 M H₂SO₄ at a flowrate of 0.4 ml per minute from a 4.6×250 mm AMINEX® HPX-87H column(Bio-Rad, Hercules, Calif., USA) at 65° C. with quantitation byintegration of glucose and cellobiose signals from refractive indexdetection using a CHEMSTATION® AGILENT® 1100 HPLC (Agilent Technologies,Santa Clara, Calif., USA) calibrated by pure sugar samples. Theresultant equivalents were used to calculate the percentage of celluloseconversion for each reaction.

The degree of cellulose conversion to glucose plus cellobiose sugars(conversion, %) was calculated using the following equation:

Conversion_((%))=(glucose+cellobiose×1.053)_((mg/ml))×100×162/(cellulose_((mg/ml))×180)=(glucose+cellobiose×1.053)_((mg/ml))×100/(cellulose_((mg/ml))×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose.

The results of the PCS hydrolysis reactions in the 50 g flask assaydescribed above are shown in Table 1. One strain that produced thehighest performing broth was designated Trichoderma reesei SaMe-MF268.

TABLE 1 Percent conversion to sugars at 168 hour timepoint Percentconversion (glucose plus cellobiose) for protein loading Broth ID-StrainName 2.5 mg/g cellulose 4.0 mg/g cellulose XCL-461-SaMe-MF268 66.2980.08 XCL-465-SaMe-MF268 69.13 82.80 XCL-462-SaMe-MF330 62.98 77.99XCL-466-SaMe-MF330 63.34 77.90 XCL-463-SaMe-MF377 64.03 78.45XCL-467-SaMe-MF377 64.19 79.06

Example 21 Preparation of Trichoderma Reesei Broths ContainingThermoascus Aurantiacus Polypeptide GH61A and Apergillus OryzaeBeta-Glucosidase Fusion Protein

Fermentation broth samples, prepared as described in Example 20, werecleared of cellular debris by centrifuging for approximately 20 minutesat 9500× g. Cleared broth samples were then filtered using a MILLEX® GPExpress™ membrane, polyethersulfone, 0.22 μm (Millipore, Bedford, Mass.,USA). The filtered broth samples were then desalted using a HIPREP™26/10 Desalting Column (AKTA™, GE Healthcare, Piscataway, N.J., USA).Protein concentrations of the desalted material were determined using aBCA™ Protein Assay Kit (Pierce, Rockford, Ill., USA) in which bovineserum albumin was used as a protein standard and calculation made forprotein in filtered broth. Aliquots were typically analyzed on 8-16%CRITERION™ SDS-PAGE gels (Bio-Rad, Hercules, Calif.; 200 V for 1 hour)in which PRECISION PLUS PROTEIN™ molecular weight standards (Bio-Rad,Hercules, Calif., USA) were included. Gels were stained for proteinusing BIO-SAFE™ Coomassie Stain (Bio-Rad, Hercules, Calif., USA) anddestained using deionized water. Estimates for the amount of Thermoascusaurantiacus GH61A polypeptide and fusion protein comprising Humicolainsolens GH45 core protein and Apergillus oryzae beta-glucosidase weremade from quantitation of scans of stained gels or by peak size afteranalysis by EXPERION™ capillary electrophoresis (Bio-Rad, Hercules,Calif., USA).

Example 22 Increase in Cellulolytic Activity of Trichoderma Reesei BrothContaining Apergillus Oryzae Beta-Glucosidase Fusion Protein whenCombined with Metal Ions and Thermoascus Aurantiacus Polypeptide GH61A

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using dilute sulfuric acid. Thefollowing conditions were used for the pretreatment: 1.4 wt % sulfuricacid at 195° C. for 4.5 minutes. According to limit digestion withexcess cellulase enzymes, the water-insoluble solids in the pretreatedcorn stover (PCS) contained 59.2% cellulose. Prior to enzymatichydrolysis, the PCS was washed with a large volume of deionized wateruntil soluble acid and sugars were removed. The dry weight of thewater-washed PCS was found to be 19.16%.

Hydrolysis of PCS was conducted using 125 ml screw-top Erlenmeyer flasks(VWR, West Chester, Pa., USA) using a total reaction mass of 50 gaccording to NREL Laboratory Analytical Protocol #008. In this protocolhydrolysis of PCS (approximately 11.3% in PCS and 6.7% cellulose inaqueous 50 mM sodium acetate pH 5.0 buffer) was performed usingdifferent protein loadings (expressed as mg of protein per gram ofcellulose) of the Trichoderma reesei fermentation broth containing theThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity and Aspergillus oryzae beta-glucosidase fusion protein with andwithout addition of 10 mM final concentration of divalent metal ions inthe form shown in Table 1. Testing of PCS hydrolyzing capability wasperformed at 50° C. with orbital shaking at 150 rpm using an INNOVA®4080 Incubator (New Brunswick Scientific, Edison, N.J., USA). Aliquotswere taken during the course of hydrolysis at 72 and 120 hours. Aliquotswere centrifuged, and the supernatant liquid was filtered using aMULTISCREEN® HV 0.45 μm membrane by centrifugation at 2000 rpm for 15minutes using a SORVALL® RT7 plate centrifuge. When not usedimmediately, filtered sugary aliquots were frozen at −20° C. Sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured afterelution by 0.005 M H₂SO₄ at a flow rate of 0.4 ml per minute from a4.6×250 mm AMINEX® HPX-87H column at 65° C. with quantitation byintegration of glucose and cellobiose signal from refractive indexdetection using a CHEMSTATION®, AGILENT® 1100 HPLC (AgilentTechnologies, Santa Clara, Calif., USA) calibrated by pure sugarsamples. The resultant equivalents were used to calculate the percentageof cellulose conversion for each reaction.

The degree of cellulose conversion to glucose plus cellobiose sugars(conversion, %) was calculated using the following equation:

%Conversion=(glucose+cellobiose×1.053)_((mg/ml))×100×162/(cellulose_((mg/ml))×180)=(glucose+cellobiose×1.053)_((mg/ml))×100/(cellulose_((mg/ml))×1.111)

In this equation the factor 1.111 reflects the weight gain in convertingcellulose to glucose, and the factor 1.053 reflects the weight gain inconverting cellobiose to glucose. Cellulose in PCS was determined by alimit digest of PCS to release glucose and cellobiose.

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at 4° C.

The results shown in Table 2 demonstrated that addition of 10 mM MnSO₄increased conversion to glucose above that obtained by equivalentamounts of fermentation broth alone at protein levels from 4 to 8 mg/gcellulose. Similarly, CaCl₂, MgCl₂, or CoCl₂ added to 10 mM increasedconversion to glucose above that obtained by equivalent amounts offermentation broth alone at protein levels from 4 to 8 mg/g cellulose.Addition of other divalent metal salts such as 10 mM ZnCl₂ or FeSO₄, orthe addition of 10 mM EDTA, a chelator of divalent cations, decreasedthe yield of glucose and cellobiose. The results for 72 hour hydrolysisare shown in FIG. 16.

The addition of 10 mM MnSO₄, CaCl₂, MgCl₂, or CoCl₂ tocellulase-containing solutions increased, therefore, the yield ofglucose and cellobiose upon hydrolysis of acid-pretreated corn stover(PCS) in comparison to addition other metals salts.

TABLE 2 Actual Actual % Actual % total Mixture of Biomass Celluloseprotein enzymes and Content Content mg/g Conversion ConversionExperiment metal (w/w) (w/w) cellulose 72 hr 120 hr 1 SaMe MF268 11.326.70 4.00 74.10 84.62 2 SaMe MF268 11.32 6.70 6.00 84.59 91.94 3 SaMeMF268 11.32 6.70 8.00 88.68 95.20 7 SaMe MF268 11.32 6.70 4.00 74.7986.14 Ca++ 8 SaMe MF268 11.32 6.70 6.00 85.77 95.11 Ca++ 9 SaMe MF26811.32 6.70 8.00 89.54 96.29 Ca++ 10 SaMe MF268 11.32 6.70 4.00 75.3187.08 Mg++ 11 SaMe MF268 11.32 6.70 6.00 86.99 95.12 Mg++ 12 SaMe MF26811.32 6.70 8.00 90.49 97.56 Mg++ 13 SaMe MF268 11.32 6.70 4.00 79.3086.77 Mn++ 14 SaMe MF268 11.33 6.70 6.00 91.65 95.23 Mn++ 15 SaMe MF26811.32 6.70 8.00 98.03 98.70 Mn++ 16 SaMe MF268 11.32 6.70 4.00 56.0063.38 Zn++ 17 SaMe MF268 11.32 6.70 6.00 65.79 75.03 Zn++ 18 SaMe MF26811.32 6.70 8.00 74.88 84.22 Zn++ 19 SaMe MF268 11.32 6.70 4.00 33.8035.83 Fe++ 20 SaMe MF268 11.32 6.70 6.00 42.50 44.88 Fe++ 21 SaMe MF26811.32 6.70 8.00 49.74 50.21 Fe++ 22 SaMe MF268 11.33 6.70 4.00 75.9885.88 Co++ 23 SaMe MF268 11.32 6.70 6.00 87.86 94.33 Co++ 24 SaMe MF26811.32 6.70 8.00 94.26 97.05 Co++ 25 SaMe MF268 11.31 6.69 4.00 57.9465.94 EDTA 26 SaMe MF268 11.32 6.70 6.00 67.67 74.35 EDTA 27 SaMe MF26811.32 6.70 8.00 72.85 77.69 EDTA

Example 23 The Increase in Cellulolytic Activity of Desalted TrichodermaReesei Broth Apergillus Oryzae Beta-Glucosidase when Combined with MetalIons is Specific to Mixtures Containing Polypeptide GH61A

The Thermoascus aurantiacus polypeptide GH61A having cellulolyticenhancing activity was recombinantly produced in Aspergillus oryzaeJaL250 according to WO 2005/074656. Fungal broth expressing Aspergillusoryzae beta-glucosidase and Trichoderma reesei cellulases wasrecombinantly produced in Trichoderma reesei strain SMA135-04.Fermentations were run for each of these proteins as described below.

For fermentations involving Aspergillus oryzae, 100 ml of a shake flaskmedium was added to a 500 ml shake flask. The shake flask medium wascomposed per liter of 50 g of sucrose, 10 g of KH₂PO₄, 0.5 g of CaCl₂, 2g of MgSO₄·7H₂O, 2 g of K₂SO₄, 2 g of urea, 10 g of yeast extract, 2 gof citric acid, and 0.5 ml of trace metals solution. Trace metalssolution was composed per liter of 13.8 g of FeSO₄·7H₂O, 14.3 g ofZnSO₄·7H₂O, 8.5 g of MnSO₄·H₂O, 2.5 g of CuSO₄·5H₂O, and 3 g of citricacid. The shake flask was inoculated with two plugs from a solid plateculture of Aspergillus oryzae and incubated at 34° C. on an orbitalshaker at 200 rpm for 24 hours. Fifty ml of the shake flask broth wasused to inoculate a 3 liter fermentation vessel containing 1.8 liters ofa fermentation batch medium composed per liter of 10 g of yeast extract,24 g of sucrose, 5 g of (NH₄)₂SO₄, 2 g of KH₂PO₄, 0.5 g of CaCl₂·2H₂O, 2g of MgSO₄.7H₂O, 1 g of citric acid, 2 g of K₂SO₄, 0.5 ml of anti-foam,and 0.5 ml of trace metals solution. Trace metals solution was composedper liter of 13.8 g of FeSO₄·7H₂O, 14.3 g of ZnSO₄·7H₂O, 8.5 g ofMnSO₄·H₂O, 2.5 g of CuSO₄·5H₂O, and 3 g of citric acid. Fermentationfeed medium was composed of maltose and antifoam. The fermentation feedmedium was dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours.The fermentation vessel was maintained at a temperature of 34° C. and pHwas controlled to a set-point of 6.1+/−0.1. Air was added to the vesselat a rate of 1 vvm and the broth was agitated by Rushton impellerrotating at 1100 to 1300 rpm.

Fermentations involving Trichoderma reesei were performed as describedin Example 20.

Crude broth samples were cleared of cellular debris by centrifuging forapproximately 20 minutes at 9500× g. Cleared broth samples were thenfiltered using a MILLEX® GP Express™ membrane, polyethersulfone, 0.22μm. The filtered broth samples were then desalted using a HIPREP™ 26/10Desalting Column. Protein concentrations of the desalted material weredetermined using a BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard and calculation made for protein infiltered broth. Aliquots were typically analyzed on 8-16% CRITERION™SDS-PAGE gels (200 V for 1 hour) in which PRECISION PLUS PROTEIN™molecular weight standards were included. Gels were stained for proteinusing BIO-SAFE™ Coomassie Stain (and destained using deionized water.Proteins were also prepared by desalting as above to remove metal ionsdissolved in fermentation broths and are indicated by the prefix ds asseen in Tables 3 and 4.

Hydrolysis of PCS was conducted using 125 ml screw-top Erlenmeyer flaskswith a total reaction mass of 50 g according to NREL LaboratoryAnalytical Protocol #008. In this protocol hydrolysis of PCS(approximately 11.3% in PCS and 6.7% cellulose in aqueous 50 mM sodiumacetate pH 5.0 buffer) was performed using constant total proteinloading (expressed as mg of enzyme per gram of cellulose) of afermentation broth containing Trichoderma reesei broth containingAspergillus oryzae beta-glucosidase with and without addition ofThermoascus aurantiacus polypeptide GH61A with and without 1 mM finalconcentration of divalent metal ions in the form shown in Table 2.Testing of PCS hydrolyzing capability was performed as described inExample 22. The degree of cellulose conversion to glucose pluscellobiose sugars (% conversion) was calculated using the equationdescribed in Example 22.

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at 4° C.

The results shown in Table 3 demonstrated that addition of 1 mM divalentcations alone did not increase glucose conversion above that obtained byequivalent amounts of fermentation broth without inclusion ofThermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity. Similarly, inclusion of the GH61A polypeptide without additionof divalent metal cations did not increase glucose conversion. Theresults for 72 hour hydrolysis are plotted in FIG. 17.

The addition of 1 mM MnSO₄ or CaCl₂ to cellulase-containing solutionswith Thermoascus aurantiacus GH61A polypeptide having cellulolyticenhancing activity increased, therefore, the yield of glucose andcellobiose upon hydrolysis of acid-pretreated corn stover (PCS). Theincrease was dependent on the presence of Thermoascus aurantiacus GH61Apolypeptide. The addition of 1 mM of other metals such as Zn++ and Fe++or chelation of metals by 1 mM EDTA reduced the yield of glucose andcellobiose upon hydrolysis of acid-pretreated corn stover (PCS).

TABLE 3 Conversion Conversion Experiment Mixture of enzymes and metal 72hr 120 hr 56 dsSMA135 54.42 62.65 58 dsSMA135 Ca++ 55.48 61.52 60dsSMA135 Mn++ 45.19 60.11 62 dsSMA135 EDTA 49.65 59.52 64 dsSMA135 Zn++37.61 43.59 65 dsSMA135 + GH61A 56.97 76.41 66 dsSMA135 + GH61A Ca++63.23 77.79 67 dsSMA135 + GH61A Mn++ 67.43 84.02 68 dsSMA135 + GH61AEDTA 49.03 57.49 69 dsSMA135 + GH61A Zn++ 47.39 58.01

Example 24 The Increase in Cellulolytic Activity of Trichoderma ReeseiBroth Containing Apergillus Oryzae Beta-Glucosidase Fusion Protein andThermoascus Aurantiacus GH61A Polypeptide when Combined with MnSO₄ andMgCl₂

Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancingactivity and a fusion protein consisting of the Humicola insolens GH45Acore with Aspergillus oryzae beta-glucosidase was recombinantly producedin Trichoderma reesei SaMe MF268 as described in Example 20.

Crude broth samples were cleared of cellular debris by centrifugingcultures for approximately 20 minutes at 9500× g. Cleared broth sampleswere then filtered using a MILLEX® GP Express™ membrane,polyethersulfone, 0.22 μm. The filtered broth samples were then desaltedusing a HIPREP™ 26/10 Desalting Column. Protein concentrations of thedesalted material were determined using a BCA™ Protein Assay Kit inwhich bovine serum albumin was used as a protein standard andcalculation made for protein in filtered broth. Aliquots were typicallyanalyzed on 8-16% CRITERION™ SDS-PAGE gels (200 V for 1 hour) in whichPRECISION PLUS PROTEIN™ molecular weight standards were included. Gelswere stained for protein using BIO-SAFE™ Coomassie Stain (and destainedusing deionized water. Proteins were also prepared by desalting as aboveto remove metal ions dissolved in fermentation broths.

Hydrolysis of PCS was conducted using 125 ml screw-top Erlenmeyer flaskswith a total reaction mass of 50 g according to NREL LaboratoryAnalytical Protocol #008. In this protocol hydrolysis of PCS(approximately 11.3% in PCS and 6.7% cellulose in aqueous 50 mM sodiumacetate pH 5.0 buffer) was performed using constant total proteinloading (expressed as mg of enzyme per gram of cellulose) of desalted orundesalted Trichoderma reesei fermentation broth containing Aspergillusoryzae beta-glucosidase fusion protein and without Thermoascusaurantiacus GH61A polypeptide with increasing final concentration ofMnSO₄ and MgCl₂ as shown in Table 4. Testing of PCS hydrolyzingcapability was performed as described in Example 22. The degree ofcellulose conversion to glucose plus cellobiose sugars (conversion, %)was calculated using the equation described in Example 22.

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at 4° C.

The results shown in Table 4 demonstrated that addition of increasingconcentration of MnSO₄ and MgCl₂ from 0.001, 0.01, 0.1, 1, or 10 mMincreased glucose conversion above that obtained by equivalent amountsof Trichoderma reesei SaMe MF268 without MnSO₄. Similarly, addition ofMnSO₄ at increasing concentrations of 0.001, 0.01, 0.1, 1, or 10 mMshowed no increase until approximately 1 mM when added to desaltedTrichoderma reesei SaMe MF268 fermentation broth. The results for 72hour hydrolysis are shown in FIG. 18.

The addition of MnSO₄ and MgCl₂ to a final concentration of 0.001, 0.01,0.1, 1, or 10 mM of each metal ion to solutions containing Trichodermareesei SaMe MF268 fermentation broth increased, therefore, the yield ofglucose and cellobiose upon hydrolysis of acid-pretreated corn stover(PCS). The addition of MnSO₄ and MgCl₂ to a final concentration of 1 mMor 10 mM to solutions containing desalted Trichoderma reesei SaMe MF268fermentation broth increased, therefore, the yield of glucose andcellobiose upon hydrolysis of acid-pretreated corn stover (PCS).

TABLE 4 Actual Actual % Actual % total Mixture of Biomass Celluloseprotein enzymes and Content Content mg/g Conversion ConversionExperiment metal (w/w) (w/w) cellulose 72 hr 120 hr 46 SaMe MF268 11.326.70 4.03 69.20 80.61 47 SaMe MF268 11.32 6.70 4.03 73.56 82.98 0.01 mMions 48 SaMe MF268 0.1 mM 11.31 6.69 4.03 74.59 84.82 ions 49 SaMe MF2681 mM 11.21 6.63 4.03 75.62 84.56 ions 50 SaMe MF268 10 mM 10.29 6.094.03 76.02 84.45 ions 51 dsSaMe MF268 11.32 6.70 4.02 70.93 79.11 52dsSaMe MF268 11.32 6.70 4.02 71.16 83.63 0.01 mM ions 53 dsSaMe MF26811.31 6.69 4.02 70.88 83.30 0.1 mM ions 54 dsSaMe MF268 11.21 6.63 4.0274.90 83.22 1 mM ions 55 dsSaMe MF268 10.29 6.09 4.02 74.17 79.01 10 mMions

Deposits of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:

Deposit Accession Number Date of Deposit E. coli strain pEJG120 NRRLB-30699 Dec. 19, 2003 E. coli strain pTter61C NRRL B-30823 Jan. 21, 2005E. coli strain pTter61D NRRL B-30812 Jan. 21, 2005 E. coli strainpTter61E NRRL B-30814 Jan. 21, 2005 E. coli strain pTter61G NRRL B-30811Jan. 21, 2005 E. coli strain pDZA2-7 NRRL B-30704 Jan. 30, 2004 E. colistrain pTr3337 NRRL B-30878 Sep. 20, 2005 E. coli TOP10 (pEJG113) NRRLB-30695 Oct. 17, 2003 E. coli TOP10 pKKAB NRRL B-30860 Jul. 8, 2005NN049573 DSM 14240 Apr. 19, 2001

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-27. (canceled)
 28. A method for degrading or converting acellulose-containing material, comprising: treating thecellulose-containing material with a cellulolytic enzyme compositioncomprising an effective amount of a polypeptide having cellulolyticenhancing activity and a soluble activating divalent metal cation,wherein the soluble activating divalent metal cation is present at aneffective concentration of about 0.001 mM to about 50 mM duringdegradation or conversion of the cellulose-containing material and thepresence of the soluble activating divalent metal cation and the GH61polypeptide having cellulolytic enhancing activity increases thedegradation or conversion of the cellulose-containing material by thecellulolytic enzyme composition compared to the GH61 polypeptide havingcellulolytic enhancing activity without the soluble activating divalentmetal cation, wherein the soluble activating divalent metal cation isselected from the group consisting of Mn⁺⁺, Co⁺⁺, Mg⁺⁺, Ca⁺⁺, and acombination thereof.
 29. The method of claim 28, wherein the solubleactivating divalent metal cation is Mn⁺⁺.
 30. The method of claim 28,wherein the soluble activating divalent metal cation is Co⁺⁺.
 31. Themethod of claim 28, wherein the soluble activating divalent metal cationis mg⁺⁺.
 32. The method of claim 28, wherein the soluble activatingdivalent metal cation is Ca⁺⁺.
 33. The method of claim 28, wherein theGH61 polypeptide having cellulolytic enhancing activity is selected fromthe group consisting of: (a) a GH61 polypeptide having cellulolyticenhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 97 or SEQID NO: 98) and [FW]-[TF]-K-[AIV] (SEQ ID NO: 104), wherein X is anyamino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, andX(4) is any amino acid at 4 contiguous positions; and (b) a GH61polypeptide having cellulolytic enhancing activity comprising[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ] (SEQ ID NO: 102 orSEQ ID NO: 103), wherein x is any amino acid, x(4,5) is any amino acidat 4 or 5 contiguous positions, and x(3) is any amino acid at 3contiguous positions; wherein the GH61 polypeptide having cellulolyticenhancing activity comprising[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] (SEQ ID NO: 97 or SEQID NO: 98) and [FW]-[TF]-K-[AIV] (SEQ ID NO: 104) optionally furthercomprises: (SEQ ID NO: 99 or SEQ ID NO: 100)H-X(1,2)-G-P-X(3)-[YW]-[AILMV], (SEQ ID NO: 101)[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or (SEQ ID NO: 99 or SEQ ID NO:100) H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and (SEQ ID NO: 101)[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions.
 34. The method ofclaim 28, wherein the GH61 polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (a) a GH61polypeptide comprising an amino acid sequence having at least 90%sequence identity to the mature polypeptide of SEQ ID NO: 12; (b) a GH61polypeptide encoded by a polynucleotide comprising a nucleotide sequencehaving at least 90% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 11; and (c) a GH61 polypeptide comprising themature polypeptide of SEQ ID NO: 12; or a fragment thereof havingcellulolytic enhancing activity.
 35. The method of claim 28, wherein theGH61 polypeptide having cellulolytic enhancing activity is encoded bythe polynucleotide contained in plasmid pDZA2-7 which is contained in E.coli NRRL B-30704.
 36. The method of claim 28, wherein the GH61polypeptide comprises an amino acid sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO:
 12. 37. Themethod of claim 36, wherein the GH61 polypeptide comprises an amino acidsequence having at least 96% sequence identity to the mature polypeptideof SEQ ID NO:
 12. 38. The method of claim 37, wherein the GH61polypeptide comprises an amino acid sequence having at least 97%sequence identity to the mature polypeptide of SEQ ID NO:
 12. 39. Themethod of claim 38, wherein the GH61 polypeptide comprises an amino acidsequence having at least 98% sequence identity to the mature polypeptideof SEQ ID NO:
 12. 40. The method of claim 39, wherein the GH61polypeptide comprises an amino acid sequence having at least 99%sequence identity to the mature polypeptide of SEQ ID NO:
 12. 41. Themethod of claim 28, wherein the GH61 polypeptide is encoded by apolynucleotide comprising a nucleotide sequence having at least 95%sequence identity to the mature polypeptide of SEQ ID NO:
 11. 42. Themethod of claim 28, where the GH61 polypeptide comprises the maturepolypeptide of SEQ ID NO: 12 or a fragment thereof having cellulolyticenhancing activity.
 43. The method of claim 28, where the GH61polypeptide consists of the mature polypeptide of SEQ ID NO:
 12. 44. Themethod of claim 28, further comprising treating the cellulose-containingmaterial with an effective amount of a beta-glucosidase.
 45. The methodof claim 28, further comprising treating the cellulose-containingmaterial with an effective amount of one or more enzymes selected fromthe group consisting of a hemicellulase, esterase, protease, laccase,peroxidase, or a mixture thereof.
 46. The method of claim 28, furthercomprising supplementing the concentration of the soluble activatingdivalent metal cation to maintain the effective concentration of thesoluble activating divalent metal cation at preferably about 0.001 mM toabout 50 mM, more preferably about 0.01 mM to about 25 mM, morepreferably about 0.1 mM to about 25 mM, more preferably about 0.1 mM toabout 10 mM, even more preferably about 0.3 mM to about 5 mM, mostpreferably about 0.3 mM to about 2.5 mM, and even most preferably about0.3 mM to about 1 mM.
 47. The method of claim 28, further comprisingrecovering the degraded or converted cellulose-containing material.